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Thank you for your resubmission and patience.
[# PeerJ Staff Note - this decision was reviewed and approved by Robert Toonen, a PeerJ Section Editor covering this Section #]
Thank you again for your resubmission. The manuscript is improved but still has a large number of English grammatical and usage errors. Please consult a professional proofreader/scientific editing service before resubmitting.
There are several other areas that can help the manuscript's clarity.
The two sentences in Lines 29-31 should include several references.
Lines 94-96 should be eliminated or worked into the Introduction
The following sentences beginning at Line 107 are not Materials and Methods and should be incorporated into the Introduction. "In a previous 108 study, the dry weight of the microalgae was correlated with the OD of the algae cultures (del Campo et al.,109 2014). Limitations of the OD technique as well as its applications to other species and another culture 110 medium were discussed by Griffiths and co-workers (Griffiths et al., 2011).
Thank you for resubmitting your manuscript. The manuscript has addressed the reviewer's scientific concerns, however, it still needs careful editing for English usage and minor punctuation/grammar mistakes. Attached are some suggested edits through p. 3. Please note that the ENTIRE manuscript needs a careful review for grammatical errors and writing in standard English.
Please consider the reviewer's comment in their entirety. The references that Reviewer 2 suggests should be included since it is important to examine the work in the context of other related scientific work. The paper is an interesting approach to characterize microalgae cell concentrations in real-time for aqua culture applications and I look forward to receiving your revised manuscript.
First, I would like to commend the authors for their work. It is a very interesting and useful research. Second, I appreciate for the raw data set available. In my opinion the article is good, but it needs an improvement on English language to ensure that it follows the professional clearly written form. Some verb tenses should be fixed to the past, which I think is the right way to describe a research done. There are also some extra commas. I suggest that you have a native English spoken colleague reviewing your manuscript.
I liked the organization of the manuscript, the objective is clear, the methods explained and discussed later. Conclusion is also understandable.
The experimental design it not well explained. The experimental design, the main hypothesis, treatments and how the data were going to be analyzed are not described.
I suggest a better explanation about the statistics analysis.
I consider the results valid although the statistic design is not well explained.
“Off-line and on-line optical monitoring of microalgal growth,” by Lazcano-Hernandez et al., describes the implementation of three optical approaches for assessing microalgal cultures based upon (1) the chlorophyll fluorescence ratio F685/F740, (2) the color of a culture backlit by a fluorescent lamp, and (3) the penetration depth of a superluminescent diode. In motivating this work, the authors state that “real time non-invasive methodologies are still needed to monitor the growth conditions of the microalgae culture.” As reviewed by Havlik et al. (“Monitoring of microalgal processes,” Adv. Biochem. Eng. Biotechnol. 153, 89-142, 2016), there have been several such methods pursued over the past decade, and I would recommend that the paper reference this relevant prior work.
As the authors state, the trends they observe in the chlorophyll fluorescence ratio F685/F740 of Chlamydomonas reinhardtii have been previously reported for plants. Also, the relationship reported between luminosity and cell concentration should be expected to be dependent upon the spectral content of the backlighting (e.g., fluorescence lamps vs. LEDs). To allow for reproduction by others, the spectral content of the fluorescence lamps should be reported. Finally, I would recommend that the authors include the results of Experiments 4-5 in Fig. 8, as graphically depicting these results may motivate others to hypothesize reasons for the lack of agreement with Experiments 1-3.
The authors have demonstrated algal biomass assessment by measuring the attenuation of light. This is somewhat routine practice by the phycology community in the measurement of OD 750 (see, e.g., the discussion by Griffiths et al., “Interference by pigment in the estimation of micoalgal biomass concentration by optical density,” J. Microbiol. Meth. 85, 119-123, 2011). Lazcano-Hernandez et al. demonstrate methods for assessing light attenuation without needing to extract a sample. A challenge, left unaddressed by the authors, is that the observed light attenuation remains dependent upon the measurement geometry, which would likely be different for every photobioreactor geometry. And because the optical methods described depend upon the culture’s absorption, each new organism would require a new assessment as well.
Related to the topic of culture absorption, the authors should wonder why the slopes in Fig. 9 are all near unity. The optical density of the culture is being measured at 640 nm, while the luminescent diode exhibits peak emission at 440 nm. Chlamydomonas reinhardtii exhibits 2-3 times more absorption at 440 nm than at 640 nm (see de Mooij et al., “Impact of light color on photobioreactor productivity,” Algal Res. 15, 32-42, 2016), so one would expect the lines in Fig. 9 to display slopes significantly greater than unity.
In the Conclusions, the authors claim that their on-line techniques could be extended to remote sensing applications. Without additional caveats, this should be clarified as speculation. Remote sensing systems typically co-locate the transmitter and receiver, which would not allow for the off-axis lighting and backlighting geometries implemented by the authors in this work.
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