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 Comments Reviewer: 1 Criteria Score: 6 The approach is reasonably good. How will multiple causes be addressed, or non-organic causes? What sort of modeling will be used. Many protocols need promulgation, i.e. sampling, storage, etc. Reviewer: 2 Criteria Score: 8 This project began in October 2010 and is 25% complete. The project partners represent broad expertise. The goal of the project is "dipstick" development of rapid detection of common algal pond pathogens and detection of "novel" problems within several days by other molecular analysis (not detailed: Hi-Seq? Cloning?). This is an important problem and the overall approach is promising, including use of biotinylated DNA and subtraction technology with microfluidics to enrich for abnormal (pathogenic) sequences. The FY11 milestones are well defined. Reviewer: 3 Criteria Score: 5 Develop methods to enrich for nucleic acids that are likely to derive from the etiological agent of the crash. Utilize ultra high throughput sequencing to identify agents. Create quantitative assays that facilitate the detection of agents at low concentration. Recreate the crash under laboratory conditions. Utilize advance spectroscopic methods to detect early hallmarks of algal stress. Reviewer: 4 Criteria Score: 8 Promising concept, but it is not clear which types of organisms the process will be able to detect--viruses, predators, and pathogens are mentioned. Apparently bacteria, fungi, and zooplankton could be identified with this method in addition to viruses. It's odd that no particular known algae pathogens are mentioned. Non-crashing ponds similar to the subject crashing ponds should be tested as controls, to estimate the false positive rate. Reviewer: 5 Criteria Score: 5 The objective is to identify agents responsible for pond crashes. The approach is to use high throughput sequencing to detect biological agents at low concentration. This approach seems technology driven rather than driven by the needs/experiences of managers of algal cultivation systems. Reviewer: 6 Criteria Score: 6 Good work except that the justification was not made as to why etiological agents are the sources of crashes. What is the evidence justifying the funds to search here for the sources of crashes relative to the disapearance, for example, if micronutrients. In terms of looking at etiological agents, though, well thought out project. Reviewer: 7 Criteria Score: 4 A good first step for this project would be to complete a failure mode and effects analysis (FMEA) on pond crashes to identify opportunities. If pond crashes have pathogenic causes with high impact, low detectability, and potential opportunities for mitigation, then the metagenomic analysis and development of advanced spectroscopy for pathogen detection would have clear value. leveraging previous work is highly desirable, but the opportunity needs to be better defined. It also appears that this project would be more efficient if it was initiated after the production system is better defined. Page 150 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. Presenter Response The proposed research has two goals. 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). We do not propose to use high throughput sequencing as an everyday detection methodology as suggested a reviewer. There are several lines of evidence that implicate biological agents in pond crashes. The detrimental effect of protozoan grazers on mass algal culture has been well documented (Lincoln et al Aquaculture 1983, 32:331-337 ). The role of other algal pathogens such as bacteria and viruses in production ponds has not be as well documented, likely because they are not readily detectable by microscopic analysis. However, in the study of natural blooms, there has been a number of recent reports of novel algal viruses and evidence of the important role that they play in the demise of a bloom (Lange et al FEMS Microbiol Rev, 2009, 33: 295-323). In addition, bacterial (Mayali and Azam J. Eukaryot Microbiol, 2004, 51:139-144) and fungal pathogens Jia et al Ecological Engineer. 2010 36:1389-1391) have also been identified that affect algae viability and may limit or contribute to the demise of blooms . Given the fact that these agents impact algal biomass levels in natural systems it is not unreasonable to expect that they can have an affect in algal production ponds (Gachon et al Trends in Plant Science, 2010, 15: 633-640; Stephens et al Trends in Plant Science 2010, 15:554-564). The strength of the our approach is that it is not limited to detection of known agents. The methods for enrichment and characterization of nucleic acids from unknown etiological agents are applicable to the broad spectrum of potential agents (viral, bacterial, fungal, protozoan) and also to the detection and quantification of invasive or "weed" algal species. There are pond crashes that are induced strictly by the physiochemical parameters (pH, temperature, nutrient levels etc). It is also clear that there are pond crashes in which biological agents play a necessary (if not sufficient) role and that the effect of these biological agents can indeed be modulated by the physiochemical parameters. Fortunately many of the physiochemical parameters can be readily measured and it has always been the goal of this project to collect and include such data in our analyses. Unfortunately, the detection and quantification of biological agents which may play a role in pond instability is not as straightforward and this is the gap that this project intends to fill. It is our intent to study both crashed and un-crashed parallel ponds with the un-crashed ponds serving as negative controls. We will also utilize pre- and post-crash samples from the same pond as a source of controls. It is also a goal of the project to isolate, when present and possible, the etiological agent of the crash, recapitulate the crash, and to determine how physiochemical parameters modulate the ability of the agent to cause a crash. In terms of positive controls, there are several known algal pathogens (various phycoviruses, bacterial agents and predators (rotifers and other protozoa). Many of these agents are already in use in our laboratories for methods development and are intended for use as positive controls. We believe that there is insufficient data publicly available to carry out the failure mode and effects analysis, suggested by one reviewer. The current inability to detect and quantify biological agents (aside from grazers) precludes the determination of the fraction of pond crashes with pathogenic causes. Finalization of a number of protocols such as those for such as sampling and storage is an early research goal and we are converging on an appropriate solution. Page 151 of 223
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Reviewer Comments - EERE

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