The research pathway in the lab can be broken down into three types of activities:

• Collection, screening and characterization of algae.

• Biochemical and physiological studies of lipid production

• Molecular biology and genetic engineering studies

There is a logic to the sequence of these activities. Researchers first identified a need to collect and identify algae that met minimal requirements for this technology. Collection and screening occurred over a seven-year period from 1980 to 1987. Once a substantial amount of information was available on the types of oil-producing algae and their capabilities, the program began to switch its emphasis to understanding the biochemistry and physiology of oil production in algae. A natural next step was to use this information to identify approaches to genetically manipulate the metabolism of algae to enhance oil production.

Algae collection efforts initially focused on shallow, inland saline habitats, particularly in western Colorado, New Mexico and Utah. The reasoning behind collecting strains from these habitats was that the strains would be adapted to at least some of the environmental conditions expected in mass culture facilities located in the southwestern U. S. (a region identified early on as a target for deployment of the technology). Organisms isolated from shallow habitats were also expected to be more tolerant to wide swings in temperature and salinity. In the meantime, subcontractors were collecting organisms from the southeastern region of the U. S. (Florida, Mississippi, and Alabama). By 1984, researchers in the program had developed improved tools and techniques for collecting and screening organisms. These included a modified rotary screening apparatus and statistically designed saline media formulations that mimicked typical brackish water conditions in the southwest. In 1985, a rapid screening test was in place for identifying high oil-producing algae. In the last years of the collection effort, the focus switched to finding algae that were tolerant to low temperature. This expanded the reach of the collection activities into the northwest. By 1987, the algae collection contained over 3,000 species.

As the collection efforts began to wind down, it became apparent that no one single species was going to be found that met all of the needs of the technology. As a result, about midway through the collection efforts, the program began studies on the biochemistry and physiology of oil production in algae in hopes of learning how to improve the performance of existing organisms. A number of ASP subcontractors struggled to identify the so-called “lipid trigger.” These studies confirmed observations that deficiencies in nitrogen could lead to an increase in the level of oil present in many species of algae. Observations of cellular structure also supported the notion of a trigger that caused rapid build up of oil droplets in the cells during periods of nitrogen depletion.

Pre-1980	1980		1994 1995 1996
Lab Studies
By 1987, over 3,000 strains of algae had been collected.
Isolation and characterization of ACCase enzyme
Genetic Engineering of Algae
expression of foreign gene in algae using protoplasts
1st successful genetic transformation of diatom
Outdoor Culture Studies and Systems Analysis
Algae Production in Wastewater Treatment
<100 sq. m. Pond Studies (CA, HI)
1000 sq. m. Pond Study (NM)
I Systems Analysis and Resource Assessment

In the end, however, the studies conducted both by NREL researchers and program subcontractors concluded that no simple trigger for lipid production exists. Instead, we found that environmental stresses like nitrogen depletion lead to inhibition of cell division, without immediately slowing down oil production. It appeared that no simple means existed for increasing oil production, without a penalty in overall productivity due to a slowing down of cell growth. The use of nutrient depletion as a means of inducing oil production may still have merit. Some experiments conducted at NREL suggested that the kinetics of cell growth and lipid accumulation are very subtle. With a better understanding of these kinetics, it may be possible to provide a net increase in total oil productivity by carefully controlling the timing of nutrient depletion and cell harvesting.

In 1986, researchers at NREL reported on the use of Si depletion as a way to increase oil levels in diatoms. They found that when Si was used up, cell division slowed down since Si is a component of the diatoms’ cell walls. In the diatom C. cryptica, the rate of oil production remained constant once Si depletion occurred, while growth rate of the cells dropped. Further studies identified two factors that seemed to be at play in this species:

1. Si-depleted cells direct newly assimilated carbon more toward lipid production and less toward carbohydrate production.

2. Si-depleted cells slowly convert non-lipid cell components to lipids.

Diatoms store carbon in lipid form or in carbohydrate form. The results of these experiments suggested that it might be possible to alter which route the cells used for storage (see schematic below):

Through the process of photosynthesis, algae cells assimilate carbon. There are numerous metabolic pathways through which the carbon can go, resulting in synthesis of whatever compounds are needed by the cell. These pathways consist of sequences of enzymes, each of which catalyzes a specific reaction. Two possible pathways for carbon are shown on the previous page. They represent the two storage forms that carbon can take.

Researchers at NREL began to look for key enzymes in the lipid synthesis pathway. These would be enzymes whose level of activity in the cell influences the rate at which oils are formed. Think of these enzymes as valves or spigots controlling the flow of carbon down the pathway. Higher enzyme activity leads to higher rates of oil production. When algae cells increase the activity of active enzymes, they are opening up the spigot to allow greater flow of carbon to oil production. Finding such critical enzymes was key to understanding the mechanisms for controlling oil production.

By 1988, researchers had shown that increases in the levels of the enzyme Acetyl CoA Carboxylase (ACCase) correlated well with lipid accumulation during Si depletion. They also showed that the increased levels correlated with increased expression of the gene encoding for this enzyme. These findings led to a focus on isolating the enzyme and cloning the gene responsible for its expression. By the end of the program, not only had researchers successfully cloned the ACCase gene, but they had also succeeded in developing the tools for expressing foreign genes in diatoms.

In the 1990s, genetic engineering had become the main focus of the program. While we have highlighted the successes of over-expressing ACCase in diatoms, other approaches were also developed for foreign gene expression—in green algae as well as in diatoms. Another interesting sideline in the research involved studies aimed at identifying key enzymes involved in the synthesis of storage carbohydrates. Instead of over-expressing these enzymes, researchers hoped to inactivate them. Returning to our “spigot” analogy, this approach was like shutting off the flow of carbon to carbohydrates, in the hopes that it would force carbon to flow down the lipid synthesis pathway (again, see the schematic on the previous page). This work led to the discovery of a unique multifunctional enzyme in the carbohydrate synthesis pathway. This enzyme and its gene were both patented by NREL in 1996.

Outdoor Testing and Systems Analysis

The first work done in earnest by DOE on demonstration of algae technology for energy production predates the Aquatic Species Program. In 1976, the Energy Research and Development Administration (before it was folded into DOE) funded a project at the University of California Berkeley’s Richmond Field Station to evaluate a combined wastewater treatment/fuel production system based on microalgae. Over the course of several years, the Richmond Field Station demonstrated techniques for algae harvesting and for control of species growing in open ponds.

By the time the Aquatic Species Program took on microalgae research, emphasis had already moved from wastewater treatment based systems to dedicated algae farm operations. From 1980 to 1987, the program funded two parallel efforts to develop large scale mass culture systems for microalgae. One effort was at the University of California, and it was based on a so-called “High Rate Pond” (HRP) design. The other effort was carried out at the University of Hawaii, where a patented “Algae

Raceway Production System” (ARPS). Both designs utilized open raceway designs. The HRP design was based on a shallow, mixed raceway concept developed at Berkeley in 1963 and successfully applied in wastewater treatment operations in California. The ARPS was really a variation on the same concept. Both efforts carried out their test work in ponds of 100 square meters or less. They studied a variety of fundamental operational issues, such as the effects of fluid flow patterns, light intensity, dissolved oxygen levels, pH and algae harvesting methods.

At the conclusion of the smaller scale tests conducted in California and Hawaii, the program engaged in a competitive bidding process to select a system design for scale up of algae mass culture. The HRP design evaluated at UC Berkeley was selected for scale-up. The “Outdoor Test Facility” (OTF) was designed and built at the site of an abandoned water treatment plant in Roswell, New Mexico. From 1988 to 1990, 1,000 square meter ponds were successfully operated at Roswell. This project demonstrated how to achieve very efficient (>90%) utilization of CO2 in large ponds. The best results were obtained using native species of algae that naturally took over in the ponds (as opposed to using laboratory cultures). The OTF also demonstrated production of high levels of oil in algae using both nitrogen and silica depletion strategies. While daily productivities did reach program target levels of 50 grams per square per day, overall productivity was much lower (around 10 grams per square meter per day) due to the number of cold temperature days encountered at this site. Nevertheless, the project established the proof-of-concept for large scale open pond operations. The facility was shut down in 1990, and has not been operated since.

A variety of other outdoor projects were funded over the course of the program, including a three-year project on algal biodiesel production conducted in Israel. In addition, research at the Georgia Institute of Technology was carried out in the late 1980s. This work consisted of a combination of experimental and computer modeling work. This project resulted in the development of the APM (Algal Pond Model), a computer modeling tool for predicting performance of outdoor pond systems.

Two types of systems analysis were conducted frequently over the course of the program—resource assessments and engineering design/cost analyses. The former addresses the following important question: how much impact can algae technology have on petroleum use within the limits of available resources? Engineering designs provide some input to this question as well, since such designs tell us something about the resource demands of the technology. These designs also tell us how much the technology will cost.

As early as 1982, the program began to study the question of resource availability for algae technology. Initial studies scoped out criteria and methodology that should be used in the assessment. In 1985, a study done for Argonne National Lab produced maps of the southwestern U. S. which showed suitable zones for algae production based on climate, land and water availability. In 1990, estimates of available CO2 supplies were completed for the first time. These estimates suggested that that there was enough waste CO2 available in the states where climate conditions were suitable to support 2 to 7 quads of fuel production annually. The cost of the CO2 was estimated to range anywhere from $9 to $90 per ton of CO2. This study did not consider any regionally specific data, but drew its conclusions from overall data on CO2 availability across a broad region. Also in 1990, a study was funded to assess land and water availability for algae technology in New Mexico. This study took a more regionally specific look at the resource question, but did so by sacrificing any

consideration of available CO2 supplies. This last study sums up the difficulties faced in these types of studies. The results obtained on resource availability are either able to provide a complete, but general, perspective on resources or they are more detailed in approach, but incomplete in the analysis of all resources.

Engineering design and cost studies have been done throughout the course of the ASP, with ever increasing realism in the design assumptions and cost estimates. The last set of cost estimates for the program was developed in 1995. These estimates showed that algal biodiesel cost would range from $1.40 to $4.40 per gallon based on current and long-term projections for the performance of the technology. Even with assumptions of $50 per ton of CO2 as a carbon credit, the cost of biodiesel never competes with the projected cost of petroleum diesel.