Reviewer Comments - EERE

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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 Too early to judge. Reviewer: 7 Presentations, posters , and being tied into other algae efforts at Sandia show strong tech transfer. Presenter Response The project team is in the process of developing stronger ties with algal pond operators. During the proposal process we obtained letters of support from Sapphire Energy inc and from Dr Ami Ben-Amotz (NBT, Seambiotic). We are seeking to build upon these initial interactions and develop others. We are also in the process of developing collaborations in the area metagenomic analysis and have partnered with a team that has submitted a letter of intent to the JGI community sequencing program on this topic. Sandia has strong internal experience and capabilities in the area of rapid detection that the project will leverage. 6. Overall Impressions Please provide an overall evaluation of the project, including strengths, weaknesses, the project approach, scope, and any other overall comments. Reviewer Comments Reviewer: 1 This is a potentially interesting project and approach. Successful implementation should be an aggressive goal for the investigators. Reviewer: 2 This is an excellent project with important goals and well-defined milestones. Appropriate metrics (e.g., speed of analysis, sensitivity of detection, efficiency of analysis, common pathogens being analyzed etc.) are under consideration in the development of molecular diptick technology. Reviewer: 3 Diagnosing the root causes of pond crashes at Sandia may provide some experimental results. The value of these results and conclusion would be limited by geographic and seasonal considerations. Meaning that microorganisms that feed on microalgae, the usual cause of pond crashes, would not be the same from location-to-location, season-to-season, or even year-to-year for the same location. Thus, the practical significance of the outcome from this effort would be limited. How could the PIs modify their effort to overcome this concern? Reviewer: 4 Potentially valuable project using novel methods. The growth of in-house cultures will probably be very important to making progress. Samples coming from various field sites may be too variable and infrequent to determine crash patterns. Reviewer: 5 This appeared to be a high-tech approach with limited applicability to real-world algal production. Page 156 of 223
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 Page 157 of 223
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Reviewer Comments - EERE

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