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.
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.
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