by David Schwartz
Dr. Jerry Brand, a PhD biologist and photosynthesis researcher, began his association with the UTEX Culture Collection of Algae in 1987, when he performed an extensive survey of the capacity for hydrogen gas production in UTEX strains. In 1994 he began studies of the causes of freeze damage in the microalgae maintained at UTEX, and has developed protocols to optimize conditions for cryogenetic storage microalgae and cyanobacteria.
Jerry Brand is Professor of Biology at the University of Texas at Austin, where he currently holds the Jack S. Josey Professorship in Energy Studies. He has been the Director of the Culture Collection of Algae (UTEX) at UT-Austin since 1998. Dr. Brand is developing DNA “barcode” information for algal strain identification and is continuing to investigate methods that maximize the number of UTEX strains that can be cryopreserved.
A.I.M. spoke with Dr. Brand recently to find out more about UTEX’s famed Culture Collection, what some people consider the algal equivalent to the Ark of the Covenant, and to hear what role it is playing in this era of algal scale up.
But first, a little refresher: One “alga.” Two, or more, “algae.” “Algal” is the adjective.
OK, let’s continue…
In the early 70’s, when you were a researcher at Indiana University studying the light reactions of photosynthesis in the green alga Chlamydomonas reinhardtii, did you have any sense that algae had the potential to become a critical energy source for this country?
I actually did, but I didn’t think of it in terms of oil production. I thought it would be more likely that once we understood the mechanism of photosynthesis, how it can capture light and split water to produce organic molecules from carbon dioxide, then we might be able to replicate key photosynthetic reactions in the test tube and thereby use artificial photosynthesis to make fuels and higher value carbon-containing compounds from carbon dioxide, water and light.
Did you continue to pursue that?
I never pursued the artificial photosynthesis angle directly. Instead I tried to understand the light-requiring reactions as they occurred in natural plant and algal photosynthesis, such as how water is split into hydrogen atoms and oxygen molecules, how electrons travel through the system and how energy is captured. That was a fashionable area of study in the 1970’s because everyone recognized its importance, but no one really understood the process in detail.
I have always been interested in applications. Two studies in particular remain of interest to those who wish to commercialize algae. One project was to look at hydrogen production in microalgae here in the Culture Collection. We surveyed hundreds of strains and published a manuscript that described the capability of a hundred strains in the Culture Collection to generate hydrogen gas.
The other project was a study of protocols that allow cryogenic preservation of algae. The reason for studying that technology is that algae will evolve in the laboratory when continuously cultivated over a period of years or decades. If an algae farmer wants to grow a strain that is particularly productive under local conditions, then he/she will want to use that identical strain every time a replacement is required. This is very similar to a grain farmer who wishes to purchase the same identical strain of seed every year. Strains of algae evolve with time when continuously grown, just as do seed crops, but algae are capable of changing more quickly since they reproduce more rapidly. Some genetic changes are likely to degrade the qualities of an alga that originally made it a desirable strain to cultivate.
If a strain of algae can be placed in liquid nitrogen (colder than any natural place on earth) under conditions that allow it to remain alive after thawing, then it can likely be maintained at that ultra-cold temperature for hundreds of years without any significant genetic change. We can cryogenically preserve hundreds of algal strains but have not been successful with others.
What potential problems with regard to genetic changes should be considered in culturing algae for scaled-up applications?
Algae that are grown outside are exposed to the ultraviolet light that is a natural component of sunlight, which causes UV-mediated mutations that may accumulate and become established in the culture over time. Mass cultures typically grow more slowly than do optimally-grown laboratory cultures, thus decreasing the rate of production of mutations. However, the high exposure to UV of outdoor cultures that are directly exposed to sunlight somewhat counteracts the moderating effect of slower growth.
Of course if one alga in a sea of trillions is altered genetically, it is unlikely that one mutant will become established in the culture, and in fact it is likely to not survive. Only a small percentage of mutations become established and of those which do become established, most are not significantly different from the non-mutated inhabitants of the culture.
However, a concern with algae in culture for long periods of time (years or decades) is that the culturing environment is almost certainly different from the conditions under which the alga was adapted in nature. This non-natural condition is likely to favor certain mutant forms, which may grow more rapidly than non-mutated forms under the artificial culturing conditions, thereby eventually taking over the entire culture. For example, the mutant strain may produce less oil in favor of more rapid growth.
A grower who orders a strain needs to know as much as possible about the strain, including information regarding its physiological and ecological characteristics, its chemical composition and its taxonomic identity. Unfortunately we and other culture collections of algae are only gradually obtaining the resources required to do the kind of screening that will be needed by the community of commercial growers. Our host institution recognizes this need and is currently developing a plan for expanding and improving the facilities for applied research and rapid screening of algal strains.
We are gradually getting DNA information from strains in the Culture Collection. A current project identifies the sequence of nucleotides in a segment of DNA called the ITS region. That segment evolves rapidly, so two cultures that originated from the same strain will likely have established a slightly different ITS DNA signature even after only a few years of remaining isolated from each other. Thus, if an established strain with a previously identified ITS signature is compared with an unknown strain that appears identical, a determination of the ITS sequence of the new strain will give a strong indication of whether or not it is identical to the established strain. Of course an identical ITS sequence does not guarantee that another segment of DNA has not changed over time. That is why cryopreservation is so important.
We are maintaining as many of our strains as we can under cryopreservation so that when we give a customer a strain, we and the customer can be assured that it is very likely to be exactly the strain that they may have received from us five years earlier.
The Culture Collection was started at Indiana University nearly sixty years ago. It came to the University of Texas at Austin in 1976, and has remained at the same location since then. It has grown from a few hundred strains to nearly 3,000 strains. The Collection includes representatives of every major group of algae, although the most represented are green algae, diatoms, and cyanobacteria.
Our actively-growing algae are kept in rooms that are temperature and light controlled. They are transferred to fresh growth media on a routine schedule so that they remain healthy. Over half of them are maintained cryogenically as well.
We also provide support services for those who are interested in commercializing algae. A popular service during the last two years has been our 2-day workshops for those interested in culturing and managing algae. In these workshops we present information on algal culturing methods, the chemical composition of algae, kind of contaminants that occur in algal cultures, various methods for measuring culture densities and growth rates, problems encountered in scale-up, etc. We also discuss the biological diversity of algae.
UTEX can provide prepared culture media that are used here to grow algae. We don’t necessarily recommend that a grower use our media to culture their algae because it’s not designed for rapid growth or culturing in large volumes. Instead, growers might use our culture media to maintain their cultures in small volumes, and then develop a medium that would allow strains of interest to grow rapidly and produce a substantial amount of a desired product.
We have a website (www.utex.org) that gives a lot of information about the Culture Collection and the services we provide.
You said you have around 3,000 strains of algae in the Collection? How does that break down?
Although we maintain nearly 3,000 different algal strains, in a few cases there are several strains of the same species. The UTEX Collection is very diverse. Every major group of algae is represented. Most are fresh water, but several hundred strains are marine algae that are found naturally in the oceans. Most of our strains are microalgae, visible only as individual organisms with a microscope. But we also maintain several hundred strains of macroalgae that are big enough to hold in your hand. Most of our strains can also be cultured over a fairly broad range of temperatures and grow well at room temperature.
One set of algae was isolated primarily from snow and prefers low temperatures; it is maintained in an illuminated refrigerator. Some of our strains of algae were isolated from high-temperature habitats, although most of them can be maintained at room temperature. The vast majority of our strains are maintained at 20 deg. C.
What percentage of the strains you ship end up in labs, vs. larger scale settings?
I would first say that over 90 percent of our algae are rarely ordered. Thus, many of our strains may have considerable unknown value. Of the total orders in the last five years, well over 50% are for practical applications, in contrast to basic research or teaching. Yet, a greater diversity of strains is ordered for teaching or for research. Those who are interested in biofuels or other practical applications generally order specific strains that are already known to have qualities of potential commercial value.
What strains have become most popular?
Green algae—Chlorophytes—are ordered more frequently than any others. Some of our Botryococcus, Chlorella, Dunaliella, Hematococcus and Nannochloris and a few others are ordered frequently, as are non-chlorophytes such as Isochrysis, Nannochloropsis, Tetraselmis, and certain cyanobacteria.
How many wild species are out there in your opinion?
That is almost impossible to even estimate. In part, it comes down to defining a species vs. a strain. Species of organisms generally are thought to be compartmentalized. One species is clearly distinct from another species according to some defined set of criteria. But that description is often problematic when applied to microorganisms. Two microalgae from different sources may appear very similar except for slight differences in one or more qualitative features such as size, color or shape. It may then be possible to find an alga that is about half-way between the other two in these qualities. So, to which of the two species does the new discovery belong, or should it be described as a new species?
It is possible to quantify differences by examining some portion of the DNA, as we and many others are currently doing. Digital data in the form of a sequence of nucleotides can be determined for a defined segment of DNA. A segment that is similar, but not identical, in all algae of interest is selected for sequencing. Each distinct species will have a unique ITS sequence. The problem is that two distinct isolates of microalgae that appear to belong to the same species will often have quite different ITS sequences. It is impractical to describe each different isolate of that species as a distinct species if its ITS sequence is any different from others because the number of different species would then be immeasurable. To avoid that problem, microorganisms with distinct DNA nucleotide sequences are described as distinct strains. Generally, when two identical-looking microalgae are isolated from different sites or from the same site at different times, they are identified as different strains because of the distinct possibility that the digital information encoded in their DNA will be somewhat different.
It is likely that many kinds of algae, especially those which can exist in forms that resist harsh conditions, are widespread in distribution.
They can be carried long distances as dust particles, attached to birds, adhering to ships and airplanes, and in many other ways. If an alga lands in a lake, for example, far removed from its site of origin, and the environment is favorable for its growth, then it may become established as a resident of the lake, identical to a population of algae in a lake from which it came. But in isolation from the other population its gradual changes in DNA content with time will not be identical with the gradual changes that occur in the population from which it was derived.
These differences can be quantified by measuring differences in DNA sequences. Then how much different must the sequences (or any other measure of difference) become before the populations are defined as separate species? The answer is arbitrary. It is much preferable to describe them as separate strains without attempting to state that they are in the same species.
The issue of change with time is not trivial. That is why it’s important to cryopreserve valuable strains when possible.
For those scaling up to algal farms, millions of acres, what do people need to know in dealing with strain evolution in the field?
I think that they should quantitatively measure critical characteristics of their algae with some frequency in order to be sure those qualities remain stable. If rate of growth or rate of synthesis of a desired product slows, for example, then one possibility is that the strain has evolved. A second possibility (probably more likely) is that a foreign strain with slightly different characteristics has invaded and overgrown the culture.
Microalgae grow rapidly, change with time and are relatively easily contaminated with foreign invaders. The large-scale grower must maintain a seed stock of the desired strain in order to restore the original strain whenever an inferior strain becomes established.
Finally, it should be noted that most current successful large-scale microalgal facilities grow natural strains of algae under conditions that are quite similar to the conditions these strains encounter in nature. Under those conditions, they are much less likely to evolve or to be displaced by invaders than are strains grown under conditions that differ significantly from their natural habitat. These problems become of more concern in genetically altered strains, but that is another story.
The Culture Collection of Algae (UTEX) can be visited at www.utex.org. Real visitors are welcome on the main campus of The University of Texas at Austin. Jerry Brand can be reached at email@example.com.