Reviewer Comments - EERE

2011 Algae Platform Review – Reviewer Comments
Reviewer Comments are direct transcripts of commentary and material provided by the Platform’s
Review Panel. They have not been edited or altered by the Biomass Program.
Reviewer: 6
I have conerns about the lack of positive controls in this project. Without those one can find exactly what
he's looking for because of a lack of controls and a narrow focus on only one variable causing a crash.
Reviewer: 7
The potential value of the project is not clear due to lack of information on pond crash stastics and lack of
definition of actions that can be taken on a commerical scale in response to pathogen identification.
Presenter Response
There are several published reports stating that predators can have severe deleterious effects on mass
cultures. It has also been determined that viruses can lead to the demise of algal blooms in natural systems
and that both bacterial and fungal pathogens can affect algal viability. It has also been acknowledged in
the literature that, taken together, this this evidence strongly suggests that biological agents have the
potential to severely impact algal production systems. It is also clear that the nature of these agents is
poorly understood and there is a need for tools for the rapid identification and detection of these agents.
This project will have two outcomes. The first is the development of a system for the rapid identification
of unknown agents by high throughput sequencing. The second is the development of a set of assays to
detect the etiological agents, once identified, at low concentration (a "dipstick" assay for example). The
the proposed system for agent identification will not be affected by geography and seasonal variation. On
the contrary, such an system would be universal and not dependent on any a priori knowledge of the
etiological agents. Rapid agent identification will allow for the timely development of the simpler
detection assays. An alternative strategy, that is under consideration, is the development of assays that
detect general classes of threats.
Whether an approach is high-tech or not is not relevant; a more salient question is whether the approach is
cost effective. The same cost drivers that affect environmental and clinical sample analysis for biodefense
are indeed relevant for algal production ponds. There is a need to minimize the cost of the initial analysis
and the resultant assays. The high tech approach taken towards sample preparation and initial
identification of pond crash agents will dramatically decrease analysis cost by minimizing reagent use and
decreasing in the per sample costs for DNA sequence analysis. By increasing the signal content of the
sample, we will decrease the amount of sequence data required to obtain a result, thus, increasing the
number of samples that can be run in a lane of sequencing.
It is clear to the research team that only a fraction of pond crashes will have a biological agent as a cause.
It has always been the intent in the project to utilize un-crashed parallel ponds as a source of negative
controls and known algal pathogens and predators as positive controls. Unfortunately the Principal
Investigator did not communicate this effectively during the oral presentation. Once a putative etiological
agent is identified, attempts will be made to isolate and propagate that agent and ultimately recapitulate
the crash (fulfilling Koch's postulates). This process will have the effect of limiting false positives and
identifying the abiotic parameters that contribute to the pond crashes. Metadata taken from the pond crash
(temperature, pH, nutrient levels etc) will also be analyzed along with the biological signature to further
understand the interplay between the agent and the environmental conditions. Identification and
manipulation of the salient environmental parameters (with and without putative biological agents) with
also facilitate the characterization of crashes that are due to solely to environmental stressors. However, it
is clear that we can not, in the scope of this project, measure all environmental parameters, and the causes
of some crashes will remain unknown.
We agree that given the limitations in obtaining significant numbers of samples from crashed ponds, in
house cultures will be important to making progress. We are modifying our approach to make more use of
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