National Science Foundation     |     Directorate for Engineering  (ENG)
Division of Chemical, Bioengineering, Environmental, & Transport Systems  (CBET)
CBET Research Highlights 
Notable Accomplishments from CBET Awards
CBET Research Highlight - Part A - Simplified Non-technical Version

1491 - Collaborative Research: Development of a Sustainable Production Platform for Renewable Petroleum Based Oils in Algae

Joseph Chappell  -  University of Kentucky, Lexington
Wayne R. Curtis  -  The Pennsylvania State University, University Park

Outcome or Accomplishment:  The research team from Pennsylvania State University and the University of Kentucky has achieved two critical goals for advancing economical production of biofuels from algae.  The Chappell lab at the University of Kentucky, has discovered and characterized the genes that make a biofuel which is essentially the same as crude oil and superior in many ways to alternative alcohol or fat-derived biofuels.  The Curtis lab at Penn State has developed photobioreactor systems and control strategies that allow the researchers to grow algae to cell concentrations that are many-fold higher than previously achieved.

Chappell Image
Images Above:
            (A)  Micrograph of the colony algae Botryococcus braunii surrounded with botryococcene oils.
            (B)  Biosynthesis of botryococcene via homologs (H-1,2,3) of the squalene synthase (S.S.) enzyme.
            (C)  Thin film algae photobioreactor.
            (D)  Online sensing of high density optical density via intensity modulated LED-photodiode.
Image Credits:  Wayne Curtis1, Joe Chappell2, Amalie Tuerk1 and Ryan Johnson1
                              1. The Pennsylvania State University, University Park
                              2. University of Kentucky, Lexington

Impact:  The ability to grow algae to higher densities and produce a biofuel that can be easily recovered and converted to gasoline greatly improves the feasibility of using algae to produce renewable energy.  This photosynthetic route to biofuels also recycles carbon dioxide back into the fuel product.

Explanation/background:  While there are many options for biofuel molecules, nearly all have limitations such as the need to use energy to distill alcohol, or the removal of water after making biodiesel from fatty acids.  In contrast, the molecule that is being produced in the research done at Penn State and the University of Kentucky has focused on has long been identified as ideal both for separation since it is a hydrocarbon that floats on water, and for conversion into gasoline.  This molecule is known to have contributed to current oil reserves when the algae that makes it (Botryococcus braunii) was apparently a dominant algae form on the planet.  However, until now, B. braunii is the only organism that is known to make this molecule.  Now that the genes have been isolated for its synthesis, there is the potential to express this gene in many different organisms to produce this biofuel.
Algae has the potential for very high yields per acre that harnesses sunlight and CO2 instead of sugar or carbohydrate that is fed to other biofuel microorganisms.  A fundamental problem in producing biofuels from algae is trying to grow algae to high densities, because as the number of algae increases, the light cannot penetrate the culture.  As a result, typical pond culture is less than 1 gram of cells per liter (99.9% water), and even more sophisticated tubes and walls of algae are hard pressed to reach 5 gram of cells per liter.  In this work, we used a vertical thin trickle film photobioreactor that resulted in algae that can grow to densities of more than 20 gram of cells per liter.  The increased cell density will lead to less water usage and lower production costs.

CBET Research Highlight - Part B - Engineering Technical Information

1491 - Collaborative Research: Development of a Sustainable Production Platform for Renewable Petroleum Based Oils in Algae

Joseph Chappell  -  University of Kentucky, Lexington
Wayne R. Curtis  -  The Pennsylvania State University, University Park

Background:  Algae cultures remain a promising route to biological production of chemicals including biofuels that does not compete directly for food-derived feedstocks such as corn-ethanol or soy-biodiesel.  Algae uses CO2 as carbon source and light as the reducing power to drive biofuel synthesis instead of alternative approaches that use a carbohydrate substrate (e.g. sugar, cellulose).  A challenge that remains for achieving economic feasibility is to efficiently utilize the available light to grow algae at a sufficiently high productivity, and to then recover the fuel product from the aqueous environment in a cost effective manner.  The target molecule of this work is C34 methylated triterpenes (botryococcenes) which are hydrocarbons produced by an algae (Figure 1A) that share biosynthetic pathways with sterols such as cholesterol in humans.  These hydrophobic oxygen-free molecules are extremely energy dense (43.8 KJ/g as compared to ethanol at 29.7 KJ/g), and have been shown to provide a very high yield of fuel when processed by current technologies for catalytic cracking of crude oil.  This work set out to discover the genes of Botryococcus braunii for genetic engineering of fuels, and explore ultra-high density algae growth to improve process engineering of algae biofuels.
In addition to achieving maximum light penetration using a trickling vertical film, the research team addressed the difficulty of nutrient feeds where stoichiometric balancing of nutrients results in cultures crashing unless a sophisticated 'feed-forward' control strategy is implemented.  Toward dealing with the highly variable environment of outdoor culturing, a cost effective method for online monitoring of algae biomass levels was developed to support the control strategy.

Results:  Based on the hypothesis of similar catalytic requirements for squalene synthase (SS), homolog genes were identified from cDNA libraries thorough molecular probes and bioinformatic parsing through mRNA sequences based on conserved motifs.  The highly unexpected result was to find that the botryococcene synthase (BS) was completely inactive on the presumed isoprene substrate, and instead required a second SS-homolog which apparently co-evolved to handle sequential enzymatic steps.  This unique enzymatic cascade is shown in Figure 1B.  The enzymes have been confirmed by both in vitro and in vivo heterologous expression in yeast.  Efforts to move these genes into other algae and microorganism species are ongoing.
A thin trickle-film photobioreactor (Figure 1C) was shown to provide ultra-high algae culture growth (>20 gDW/L).  the research team confirmed a hypothesis that once high-density is achieved, light-limited growth conditions occur where the productivity of a photobioreactor may no longer dependent on the intrinsic algae doubling time.  The same biomass productivity was achieved for Chlorella (which can double in 4 hours) as B. braunii (which can take 5 days or more to double).  Even more surprising was the result that B. braunii captured nearly double the total energy (bomb calorimitry) and produced substantially more biofuel than Chlorella.  Continuous operation for months revealed complex instabilities associated with CO2 transport, pH and nutrient addition, even within a controlled laboratory setting.  To achieve stable photobioreactor operation in a highly variable outdoor environment, a feed-forward / predict-correct strategy is being developed based on growth kinetic models and nutrient mass balances.  An online optical density (OD) sensor capable of measuring OD550=40+ without dilution is being validated with computer controlled nutrient addition.

- 1 -   Identification of unique mechanisms for triterpene biosynthesis in Botryococcus braunii.  Niehaus TD, Okada S, Devarenne TP, Watt DS, Sviripa V, Chappell J.; Proc Natl Acad Sci U S A. 2011 Jul 26;108(30):12260-5. Epub 2011 Jul 11.
- 2 -   Functional identification of triterpene methyltransferases from Botryococcus braunii race B.  Niehaus T, Kinison S, Okada S, Yeo YS, Bell SA, Cui P, Devarenne TP, Chappell J.; J Biol Chem. 2012, 287, 8163-8173.
- 3 -   A bioprocessing comparison of high density Botryococcus braunii and Chlorella vulgaris verifying light limited growth.  Grady, L.K., MS Thesis, The Pennsylvania State University, 69pp, May 2010. (should be released electronically March 2012)
- 4 -   An Assessment of photosynthetic biofuels and electrofuels technologies under rate-limited conditions.  Tuerk, A., MS Thesis, The Pennsylvania State University, 303pp, Dec. 2011.

Scientific Uniqueness:  Metabolic engineering for a new superior biofuel molecule has been enabled.  A photo-bioreactor design and operational strategy for ultra-high density continuous culture of algae has been developed.

This project addresses the CBET Strategic Outcome Goals as follows:
Primary Strategic Outcome Goal:      (1) Discovery:  The multi-disciplinary emphasis of NSF enabled this collaborative research team to synergistically make scientific and engineering advances related to algae biofuels.
Secondary Strategic Outcome Goal:  (2) Learning:  Over 20 students have been directly involved in the execution of this research including (2) molecular biology and (4) Chemical Engineering graduate students.  A list of these students with biosketches of their contributions can be found at this website:
The work has included assistance to middle-school curriculum development and helping a student with a high-school science fair project.  Hundreds of undergraduates have been taught a case study on algae biofuels as noted under broader impacts.  The work was executed with exchange visits of students and facilitated through bi-weekly conference calls which provided for extensive interaction and mutual appreciation of the science and engineering efforts.

Transformative Research:  The superiority of the biofuel in this work has been rapidly recognized.  As a result of the discovery of these genes for botryococcene biosynthesis, 10s of millions of additional funding has been awarded within the last 2 years by USDA, DOE, ARPA-E to examine alternative hosts (plants, microbes) to produce this biofuel.  The demonstration of photon-limited productivity has clarified a long-standing misconception where millions of dollars have (and continue) to be focusing on achieving rapid growing algae without understanding the impact under bioprocessing conditions required for fuel production.

Intellectual Merit:  The metabolic pathway discoveries illustrated the unexpected complexity of biology where presumed simple metabolic pathway is not the basis of biosynthesis of this molecule.  Similarly, the presumed simplicity of high-density algae culture proved to be a very complex interaction of mass transfer, light scattering, stoichiometry and CO2-dependent nitrogen assimilation.  This work validated the strength of interdisciplinary collaborative research.

The Broader Impacts of this research include:
- 1Benefits to society:  This research will very likely have profound impact on the field of biofuels production by introducing what may well be the ultimate biofuel molecule for liquid fuels.  The advances in photobioreactor design and operation will improve future feasibility of using algae to produce useful biochemicals for society.
- 2Broadening the participation of underrepresented groups:  Three female student carried out the majority of the bioreactor research with the assistance of over a dozen undergraduates - providing them with mentoring and leadership skills.  One of these graduate students has gone one to pursue an MD, another is pursuing a PhD on an NSF Graduate Fellowship.  The REU supplement helped to support (2) female undergraduates and the research effort was supplemented with participation in a Penn State NSF-REU Site on Energy Storage and Conversion and endowment funds to support undergraduate research.
- 3Advancing discovery and understanding while promoting teaching, training, and learning:  Undergraduates have received over 30 credits for undergraduate research work conducted under this project.  One of the graduate students (Tuerk) returned from the pharmaceutical industry to help teach a newly developed 150-student course on introduction to biomolecular engineering.  A case study on photosynthetic production systems was added to an existing biochemical Engineering class, and homework/test questions were derived from a greater understanding of the kinetics of isoprene metabolism.  One student intern came from IIT Dehli, and another from New Zealand satisfied his BS Engineering practicum requirement assisting with this project.  Additional REU students participated from Kansas State, Miami University of Ohio, and Rose-Hulman.
- 4Results disseminated broadly to enhance scientific and technological understanding:  In addition to key publications of botryococcene biosynthesis (2) MS are written with (2) more being completed and will be available electronically from the PSU library (several undergraduate thesis, posters and presentations are also available online).  Numerous publications will be forth-coming and the results have been enthusiastically received at numerous recent professional meetings.  A patent on photobioreactor technology was filed and is the basis of a startup commercialization effort.  A Hispanic undergraduate (Venezuela national) student received a 3rd place in the AIChE 2011 National Paper competition based on his algae research.

Program Director:
Theresa Good
CBET Program Director - Biotechnology, Biochemical, and Biomass Engineering
NSF Award Number:   (1)  0828817
(2)  0828648
Award Title:   Collaborative Research: Development of a Sustainable Production Platform for Renewable Petroleum Based Oils in Algae
Principal Investigators:   (1Joseph Chappell
(2Wayne R. Curtis
Institution Name:   (1)  University of Kentucky, Lexington
(2)  The Pennsylvania State University, University Park
Program Element Code:   1491
CBET Research Highlight:   Fiscal Year 2012
Approved by CBET on:   27 March 2012

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This CBET Research Highlight was Updated on 27 November 2012.