Algae Biofuels

Recent Publications

  1. Wang, J., Rosov, T., Wensel, P., McGowen, J., Curtis, W.R. (2016) A preliminary implementation of metabolic-based pH control to reduce CO2 usage in outdoor flat-panel photobioreactor cultivation of Nannochloropsis oceanica microalgae. Algal Research, 18: 288-295. DOI:10.1016/j.algal.2016.07.001

  2. Wang, Jun, Curtis, Wayne R. (2015) Proton stochiometric imbalance during algae photosynthetic growth on various nitrogen sources: Toward metabolic pH control. Journal of Applied Phycology. DOI: 10.1007/s10811-015-0551-3

  3. Khatri W, Hendrix R, Niehaus T, Chappell J, Curtis WR (2013) Hydrocarbon Production in High Density Botryococcus braunii Race B Continuous Culture, Biotechnology & Bioengineering, 111 (3), 493-503 doi:10.1002/bit.25126

  4. Myers JA, Curtis BS, Curtis WR (2013) Improving accuracy of cell and chromophore concentration measurements using optical density. BMC Biophysics 2013, 6:4, 1-16.

  5. Scherholz ML, Curtis WR. (2013) Achieving pH control in microalgal cultures through fed-batch addition of stoichiometrically-balanced growth media. BMC Biotechnol. 13(1):39, 1-16.

  6. Curtis WR, Curtis MS (2014) Biomass-2-Energy, Chapter 3, In: Systems Engineering for Clean and Renewable Energy Manufacturing in Europe and Asia, NSF sponsored Report, WTEC. (Full Report on Web; http://wtec.org/SEEM/)

Research Overview

With a rising global population and dwindling oil reserves, finding alternative sources to foreign oil, which are competitive economically yet also environmentally sustainable, is a huge priority. Algae biofuels shows particular promise in its capability for Greenhouse Gas (GHG) capture and its avoidance of the 'Food versus Fuels' paradigm in which production is limited by land space due to direct competition for food, which again will only be exacerbated by a growing population. However, harvesting algae for fuel production does come with its own unique set of obstacles to overcome.

The algae biotechnology efforts of the Curtis Lab started in earnest in ~2005 to assist the first major algal biofuels company, GreenFuel Technologies, including having several of their earliest employees undertake training at Penn State as they setup laboratories in Boston. This work also included producing 100 liters of innoculum for the MIT cogeneration powerplant demonstration. In 2007-08, Dr. Curtis went on sabbatical, devoting half of it as a technical advisor to Green Fuels Technologies startup effort in Boston.

Since then, CurtisLab has similarly continued applying chemical engineering principles to develop methods of producing algae biofuels emphasizing economic feasibility. This includes:

  • Demonstration of ultra high-density ( > 20g/L), continuous growth vis-a-vis Wayne's Freshwater Algae Media (WFAM) and a low-cost Trickle Screen Reactor (patent pending), which resulted in the Chappell Lab & CurtisLab's NSF-CBET highlight

  • Subsequent NSF-SBIR (Small Business Innovation Research) grant for Dr. Curtis' short-lived Calyx Bioprocess Technology LLC for scale-up

  • Comparison of algal biofuels candidates Botryococcus braunii and Chlorella vulgaris via growth kinetics and bomb calorimetry to substantiate B. braunii's superiority as a biofuels candidate

  • Feed-forward pH control strategies to reduce operational costs and optimize growth

  • Genetic engineering of B. braunii's botryococcene-synthesizing genes into alternative hosts

  • Novel method for maintaining laboratory-scale axenic algal cultures

  • Isolation and genetic characterization of a novel ectosymbiont

  • Curation of an open-source wiki page, botryococcus.org, to centralize information relevant to B. braunii

  • Tandem production of high-value protein and biofuel in microalgae

Photobioreactor (PBR) Design and Operational strategies

Trickle Film Reactor for Pilot-Scale Production

Many processes and photobioreactor designs in the algae community have obvious or hidden limitations that have significant impact when evaluated from a biochemical engineering stance (i.e. simple mass and energy balance principles). Alternatively, CurtisLab's Trickle Film Reactor (TFR) considers the entire algae biomass production process to address the inherent limitations of light physics, algae physiology, and economics, thereby achieving high-density growth at minimal costs, both fiscal and energetic, and limited only by light. Specifically, the TFR achieves maximum productivity via a turbulent-mixed film at minimum thickness in conjunction with light-limited, high-density algae growth. Furthermore, it facilitates low-cost downstream dewatering and reduced ‘hydraulic load’ costs throughout the process.

LEFT: Schematic of the trickle-film photobioreactor, illustrating the overall flow pattern for liquid and gas (A), a close up of the trickling film depicting the short path length for light penetration as well as mixing induced in the falling film due to drag (B), and an idealized view of how the diagonal screen increases resistance to the fluid flow path and induces vorticity to the flowing algae culture (C). RIGHT: presentation by Ryan Johnson on Calyx LLC. scale-up of Trickle-Screen Reactor.

Algae is grown as a suspension that trickles down a screen to induce turbulent mixing in a simple geometry that promotes efficient photon use by avoiding a surface that can foul between the algae and the light, thereby supporting densities of 30+g/L biomass—correlating the highest oil productivities found in literature irrespective of the intrinsic growth rate among various strains of algae. We have demonstrated that CO2 fertilization can be accomplished in the evaporative water make-up saturated with nearly pure CO2. This avoids prohibitive costs of heat removal and gas compression on agricultural scales. This approach also avoids the capital expense and safety/permitting issues of light blocking ‘green-house’ enclosures and use of existing scrubbing technologies to provide concentrated CO2.

Photos from Calyx LLC start-up effort located in rural Port Royal, PA at the Curtis Family Farm. CEO Wayne Curtis; COO: Ryan Johnson, CTO: Waqas Khatri; other s include Chelsea Berri, Robert Hendrix, Brandon Curtis. Dr. Curtis, Sr., appropriately a PSU agricultural education Ph.D., is also pictured.

Dynamic Feeding & Feed-Forward pH control

Critical to algae biofuels are operational costs associated with controlling pH, which inherently affects algal health and growth. Algae pH is affected by both carbon and nitrogen utilization, and significantly by the source of nitrogen. For example, preferntial consumption of ammonium (NH4) results in a dissociation into ammonia (NH3) and proton (H+) which lowers pH while consumption of nitrate dissociates to form nitrite (NO2) and a hydroxyl group (OH-), in turn raising pH. Unsophisticated methods of buffering that are heavily relied upon in the world of algae biofuels include dissolving CO2 to buffer these pH changes, however, they are intensively wasteful by ignoring Henry's Law for the dissolution of gasses to waste ~90% of CO2 at $50/ton and thereby antithetically contributing to GHG emissions. In revealing these significant obstacles, CurtisLab developed an OD sensor (patent pending).

Laboratory scale pH monitoring and dynamic feeding implementation

To improve this problem, CO2 was appropriately minimized to a volume appropriate for photosynthetic growth. Meanwhile, a dynamic feeding strategy was used to counteract algae's preferential consumption of ammonium---to the point of gluttonously killing itself. Finally, feed-forward process control, whereby proactive predictive modeling rather than a lagging reactive response informs the control strategy, was implemented to regulate pH. Specifically, growth predictions based on light cycles dictated the respective dosing of acids and bases to maintain pH.

This was initially demonstrated at the laboratory scale, and to date has been scaled-up for the single cell, saltwater algae, Nanochloropsis, at Arizona State's (ASU) AzCATI, the Arizona Center for Algae Technology and Innovation. Now graduated, the Ph.D. who pursued this work, Jun Wang, continues to collaborate with CurtisLab and AzCATI to improve feed-forward modeling and process control in addition to scaling and automating this process for other alga including but not limited to Hamaetococcus, Chlorella vulgaris, Galdieria sulphuria, Botryococcus braunii, Poryphridium purpureum, and Scenedesmus dimorphous, and Chlamydomonas reinhardii.

Dr. Curtis pictured with ATP3's array of algae's in indoor tubular reactors.

'Alginator' for Bench-Scale Microalgae Characterization Based on Photoefficiency Metrics

In response to the Trickle Film (TF) reactor's success in demonstrating performance of microalgae biofuels candidates under commercially relevant conditions at pilot-scale, in turn contradicting the utility of intrinsic growth rate as a valuable performance metric, the 'Alginator' bioreactor was designed as a bench-scale reactor to evaluate microalgae biofuels' performance using photoefficiency. The emphasis on efficiency of solar-to-biomass conversion is central to the Alginator's design and relevance to commercially-revelant conditions of continuous operation and high-density, light-limited growth. In principle, the Alginator is a flat-panel, external loop airlift photobioreactor that is designed to be low-cost and bench-scale to make the technology to characterize algae or, more specifically to 'bioprospect' for a biofuels production candidate, accessible for standard laboratories. The critical design elements that enable measurement of photoefficiency is the Alginator's flat-panel design, narrow path length, and defined light path & spectra. Together, these elements define the energy inputs and outputs for generating an energy balance and calculating photoefficiency. The airlift design with CO2 supplementation also promotes sufficient mixing and carbon mass transfer to sustain high-density photoautotrophic growth as shown by the dense green culture below.

Left to Right: Visiting Scholar Ramya Mohandass (SRM University, India), Ben Geveke, Lucas Nugent, and (Michael) Grant Gill in summer 2016 with Alginator.

(Note: this is one of the last laboratory photos taken in 232 Fenske Laboratory.)

Towards the Alginator's accessibility, below is a public google Drive folder with CAD drawings for Alginator design.

Bioremediation

Algae's potential not only to sequester carbon to reduce GHG emissions but also its capacity for growth on wastewater, in turn utilizing nitrogen and reducing toxicity, represents a significant advantage in the hunt for renewable fuels. This is further detailed in CurtisLab's 'Environmental' work.

Genetic Engineering for Fuels Production

Our collaborators at Chappell Lab elucidated the two biosynthetic pathways in Botryococcus braunii, the Mevalonic Acid (MVA) and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways. As part of CurtisLab's work in metabolic engineering, we engineered the botryoccene-synthesizing genes into a bacterial host as part of an ARPA-e Electrofuels grant.

Tandem Protein Production

In 2014, the Department of Energy's Bioenergy Technologies Office (DOE BETO) announced its aim to reduce algal biofuels price from its ~$8 gge (gas gallon equivalent) to $3 gge by 2030 with an intermediate goal of achieving $5 gge by 2019. To that end, algal biofuels are being pushed as a dual production platform in which the cost of producing large amounts of algal biofuels is offset by a co-produced high-value product, and significantly, one which maintains its production value at the associated production levels. Parlaying our work in using plants as a protein production platform, our ongoing work similarly aims to produce a high-value protein with the potential to be as a bioplastic derivative. CurtisLab is currently investigating the optimal platform and protein expression system.

To that end, we have previously collaborated with the Demirel Lab (PSU, Materials Sciences) to scale-up production of a potentially high-value protein for their material characterization. Further we have ongoing Penn State collaborations with the Bryant Lab (Biochemistry & Molecular Biology) as well as Phil Savage's Lab (Chemical Engineering). Future work may also include transplastomic engineering.

Establishing & Maintaining Axenic Botryococcus braunii

Akin to our TSReactor work, we also grew Botryococcus braunii semi-continuously at high-density at laboratory scale. A surprising struggle we found was in isolating Botryococcus braunii to establish axenic (i.e. B. braunii and ONLY B. braunii) cultures---in fact, taking almost a year-and-a-half to isolate it! This led to (1) a method of maintaining B. braunii to reduce contamination and (2) genomic study of the novel contaminating ectosymbiont.

Novel Axenic Maintenance Method

The video below details the SOP for maintaining a co-culture of axenic phototrophically-grown B. braunii and heterotrophically-grown plant roots for optimal gas exchange, respectively via photosynthesis and aerobic respiration. Ongoing work aims to quantify growth of numerous algae and plant root species, in turn, enabling optimized pairings of algae and plants.

Characterization of a Novel Ectosymbiont

Due to our initial difficulty isolating B. braunii, CurtisLab began to tangentially investigate the potential symbiotic relationship between this persistent bacteria and its algal host. After initial shotgun sequencing we consulted with Penn State's 'in-house' PSU Bioinformaticist expert Istvan Albert but were still left with 200 contigs. Thus, we used the new OpGen technology, a method of quite literally 'optical mapping’ which provides a unique templating method based on microscopic images of DNA stretched out onto slides, to assemble a complete genome. Ongoing analysis has revealed numerous surprises in the ectosymbiont's genome, which will soon be detailed in a paper. Additional ongoing work includes various growth studies and assays to elucidate this organism's potential as a symbiont or pathogen.

Botryococcus.Org

In our study of B. braunii as a platform for an algal biofuels candidate, it became apparent how disjointed and convoluted the literature was on B. braunii---particularly that related to hydrocarbon productivities--and to our surprise, after decades of promising research on this algae, no one has centralized this information---or at least not digitally. Therefore, in true academic spirit, CurtisLab created botryococcus.org, an open-source wiki page, for sharing all things Botryococcus. To date, our open-source 'literature review' has revealed more than 150 B. braunii strains from all around the world and found a huge range in specific productivities.

Botryococcus Citizen Science Kits

Most of the research done on the B race (most oil-rich) of Botryococcus braunii is done on the Showa strain found at UC-Berkeley in 1980. Given our surprise at the vast regions where B. braunii has been isolated and the persistence of the ectosymbiont we isolated from B. braunii, we are investigating whether this is a naturally occurring symbiosis in nature or one which has been introduced over decades of successive laboratory culturing. To that end, we are developing a Citizen Science kit which will facilitate collection of B. braunii, largely free from other algae, from all over the world to be studied in our lab and shared with the world. Specifically, we will look at using metagenomics, sometimes called community genomics in which genomic analysis of microbial DNA of an environmental sample enables a survey of different microorganisms, to investigate the organisms found naturally with this significant algae.

Grants

NSF: Petroleum-Based Algal Oils

NSF Grant Site (0828648)

Algal Biofuels Team

CurtisLab

Graduates: Lisa Grady, Waqas Khatri, Amalie Tuerk*, Jun Wang

Undergraduates: Ryan Benoit, Chris Colona, Erik Curtis, Ben Geveke, Michael Grant Gill, Nate Hamaker, Robert Hendrix, Patrick Hillery, Katie Legenski, Erica Lennox, Taylor Maher, David Martino, Mainara Muhl, Bill Muzika, Adam Nebzydoski, Marcia Rodrigues, Megerle Scherholz, Eva Mei Shouse, Steve Tran, Amalie Tuerk*,Haonan Xu, Justin Yoo, Joy Yuan

Technicians: Ryan Johnson

Chappell Lab (University of Kentucky)

Graduate Students: Tom Niehaus, Steve Gaubauer, Erik Nybo

Technician: Scott Kinison .

DOE Arizona Testbed Public-Private Partnership (ATP3)

Director: John McGowen

Technicians: Pierre Wensel, Theresa Rosov

Collaborators: Joe Chappell! (the legend =), Phillip Savage, Randy van der Waal, Don Bryant, Istvan Albert,

Other Products:

Papers / Presentations / Patents:

Curtis WR, Wang J, Khatri W, Tuerk A. A Decade of algae bioprocess engineering: the neglected importance of operational strategy and control. American Chemical Society (ACS) National meeting, Aug 16-20, 2015. Boston, MA.

Curtis WR, Wang J. Metabolism based feed forward pH control of high-density microalgae cultures. DOE-BIOMASS 2014, July 29-30, Washington, DC.

Wang J, Geveke B, Johnson R, Curtis WR. Feed-Forward Control of Dynamic High-Density Microalgae Cultures Using Model Based Predictive pH Control and a Novel Biomass Sensor. AIChE Annual meeting, November 3-8, 2013. San Francisco, CA.

Link D, Tuerk A. Overall Energy Considerations for Algae Species Comparison and Selection in Algae-to-Fuels Processes. Alternative Energy NOW Conference, Lake Buena Vista, FL, February 23, 2011.

Johnson, Ryan, Khatri, W., Curtis, W. (Calyx Bioprocess Technology, LLC., Port Royal, PA) SBIR Phase I: Hydro-ThermAlgae, Photobioreactor for Minimal Water Loss. Final Proposal [not accepted] Arlington, VA: National Science Foundation; 2012 Sep. IIP-Small Business Phase I.

Amalie Tuerk*, Wayne R. Curtis. Algae-Based Hydrocarbon Production. USDA Research Center, Philadelphia, PA, April 2010.

Curtis WR. Trickle-film Bioreactor for Growth of Photo-hetrotrophic anaerobes for Membrane protein production and growth of photosynthetic algae. Patent US 14/729,692. Applied: 2009.

Algae-Based Hydrocarbon Production

Waqas Khatri, Steve Gabauer, Joseph Chappell, Wayne Curtis. AIChE Annual Meeting, Philadelphia, PA, Nov. 18 2008

Genetic Engineering Hydrocarbon Production in Algae

Stephen Gabauer, Thomas D. Niehaus, Waqas Khatri, Joseph Chappell, Wayne Curtis. AIChE Annual Meeting, Philadelphia, PA, Nov 19 2008

Theses

Wang, Jun. Improving Process Efficiency of Algae-Based Biofuel and Bioproduct Production Using Metabolism-Based pH Control. Dissertation, Ph.D. Penn State University, University Park, PA (2015).

Yoo, Justin. Establishment and Maintenance of Axenic Botryococcus braunii race B Algae Culture. Dissertation. Penn State University: University Park, PA (2014).

Taylor, Christine. A Continuous Bioreactor to Study the Persistence of Extracellular Biofuel Candidate Molecules in a Non-axenic Algae Culture. Dissertation. Penn State University: University Park, PA (2013).

Nebzydoski, Adam. Photobioreactor Control Algorithms in LabVIEW Utilizing Algae Bag Reactors. Dissertation, B.S. The Pennsylvania State University: University Park, PA (2011).

Tuerk, Amalie. An Assessment of Photosynthetic Biofuels and Electrofuels Technologies under Rate-Limited Conditions. Dissertation, Ph.D. Penn State University: University Park, PA (2005).

Grady, Lisa. A bioprocessing comparison of high-density Botryococcus braunii and Chlorella vulgaris verifying light-limited growth. Dissertation, M.S. Penn State University, University Park, PA (2015).