Nutrient Recovery from Anaerobically Treated Blackwater and Improving Its Effluent Quality through Microalgae Biomass Production
Round 1
Reviewer 1 Report
The manuscript “Nutrient recovery from anaerobically treated blackwater and improving its effluent quality through microalgae biomass production” presents investigations conducted to assess the nutrient removal efficiency of C. sorokiniana and its potential to improve effluent quality: anaerobically digested blackwater. The manuscript contains interesting information but the paper is not excellent enough to be published in Water in this form. The manuscript contains many errors. There is no statistical analysis in the manuscript. Were the experiments performed in biological replications?
Why do the authors use such high lighting conditions in the culture: 1450 µmol photons m-2 s-1? Please provide an explanation in the manuscript (the goal intended to be achieved by the authors while using such conditions) for the use of such lighting conditions in the culture. Optimal light intensity for most species is definitely lower. What was the purpose of using continuous lighting instead of a photoperiod? Did the Authors want to induce stress conditions? In the manuscript, the authors present many data, which are unfortunately very chaotically described and this makes it difficult to interpret the research results and read the text. Throughout a great part of the text, the Authors do not provide a reference (mainly to Table 2) informing where in the manuscript the data are shown. In Results chapter 3.1, the Authors write in line 185 “showed a high specific growth rate”. There are no results of the growth rate in the manuscript. On what grounds do the authors formulate such a conclusion? The growth rate results should be added and the sentence should be reedited. The Authors report that algal growth was monitored using spectrophotometric measurements of OD but there are no results of the OD measurement in the manuscript. The Authors used dry biomass weight for measurements of PV and PA. What was the OD measurement used for?There are many errors in the manuscript, but only some are listed below. Before re-submission of the manuscript to the journal, the Authors should carefully revise their manuscript.
Line 186 page 6: “1.58±0.21” - there is no unit
Figure 2 – the abbreviation dBMR is not explained
Line 196 page 6: the Authors write that Nr is the “NO3-N removal rate”, whereas on the previous page they write that Nr is “the sum of NH4-N, NO2-N and NO3-N”
Line 196-199 p 6: information referring the results to Table 2 should be added.
The presentation of the result should be standardized: the results in Table 2 are presented to the second decimal place, but rounded in the description in the manuscript. For example, line 199 p 6: the number in the description is 5.67 whereas in Table 2 it is 5.7. There is no ± symbol in this place. The Authors do not specify the meaning of “±”- standard deviation?
Line 198 p 6: Pr is 29.5±4.1, whereas the number in Table 2 is 29±4.2
Fig 4 – there is no explanation of dBM
In the manuscript, different notations of the same units are used, e.g. mg/L or mg L-1
Author Response
Point 1
The manuscript “Nutrient recovery from anaerobically treated blackwater and improving its effluent quality through microalgae biomass production” presents investigations conducted to assess the nutrient removal efficiency of C. sorokiniana and its potential to improve effluent quality of anaerobically digested blackwater. The manuscript contains interesting information but the paper is not excellent enough to be published in Water in this form. The manuscript contains many errors.
Response 1
The errors have been corrected thoughout the manuscript accordingly.
Point 2
There is no statistical analysis in the manuscript. Were the experiments performed in biological replications?
Response 2
There were running 2 parallel chemostats and with continuous cultures also time as a factor for backing up data (steady-state over a certain time). The basic features of the data set were described using descriptive statistics and average and standard deviation (±) are reported. Wherever necessary, unstacked one way ANOVA was used to measure the overall variation between treatments. An alpha level of 0.05 used to determine statistical significance for the analyses. This is included in the material and method as section 4. Statistical analysis. Page 6, Line 180
Point 3
Why do the authors use such high lighting conditions in the culture: 1450 µmol photons m-2 s-1? Please provide an explanation in the manuscript (the goal intended to be achieved by the authors while using such conditions) for the use of such lighting conditions in the culture. Optimal light intensity for most species is definitely lower. What was the purpose of using continuous lighting instead of a photoperiod? Did the Authors want to induce stress conditions?
Response 3
There is not an optimal light intensity for a whole culture without taking the set-up of the culture system into account. There may be an optimal light intensity for one cell but in a whole culture the light intensity that one cell experiences is strongly dependent on the overall set-up, i.e. the incident light intensity, the light path, the amount of mixing, the shape of the bioreactor, and of course the biomass concentration in the bioreactor. In our case the aim was to remove as much nitrogen and phosphorous from the treated black water as possible, which resulted in high cell densities, and to avoid light limitation we chose the highest possible light intensity of our light source (LED panel) and no dark periods. For even higher nutrient concentrations (20 % concentrated treated black water was the maximum in the presented experiments) the light intensity would have to be increased even more or the light would limit the uptake of the nutrients. Just to give a perspective: In another paper (Tuantet2014, cited in our manuscript) they use an even smaller light path (10 mm) and illuminated with even higher light intensity (1530 µmol/m2/s) from both sides of the flat panel photobioreactor, resulting in a more than 6 times higher light intensity per volume of culture compared to our set-up.
Ponit 4
Throughout a great part of the text, the Authors do not provide a reference (mainly to Table 2) informing where in the manuscript the data are shown.
Response 4
Table 2 is only a summary of the data presented in the text. Although no references needed, references to this table is now included in the text.
Point 5
In Results chapter 3.1, the Authors write in line 185 “showed a high specific growth rate”. There are no results of the growth rate in the manuscript. On what grounds do the authors formulate such a conclusion? The growth rate results should be added and the sentence should be reedited.
Response 5
The specific growth rate was mentioned from the nature of the exponential phase of the graph as shown in figure 2. According to the figure, the growth of C. sorokiniana started immediately without a clear lagging phase and the fast growth shown in the exponential phase. The dry biomass concentration reached 1.72 g L-1 with in 30 hr from an initial concentration of 0.12 g L-1 in a continuous mode at a dilution rate of 0.06 h-1. It was based on this data we stated the “a high specific gravity“. However, the statement in the manuscript is now rephrased accordingly. Page 6, Line 189-192
Point 6
The Authors report that algal growth was monitored using spectrophotometric measurements of OD but there are no results of the OD measurement in the manuscript. The Authors used dry biomass weight for measurements of PV and PA. What was the OD measurement used for?
Response 6
Both OD and dry biomass concentration were measured throughout the experiment. The results were highly correlated and we chose to only use the dry biomass concentration as a direct measure of volumetric biomass productivity (PV) and areal biomass productivity (PA). The text has been rephrased accordingly.
Point 7
There are many errors in the manuscript, but only some are listed below. Before re-submission of the manuscript to the journal, the Authors should carefully revise their manuscript.
Response 7
The errors have been corrected accordingly.
Point 8
Line 186 page 6: “1.58±0.21” - there is no unit
Response 8
Unit added. 58 ± 0.21 g L-1 Page 6 , Line 192
Point 9
Figure 2 – the abbreviation dBMR is not explained
Response 9
dBMR is replaced with “X“ for dry biomass concentration
Point 10
Line 196 page 6: the Authors write that Nr is the “NO3-N removal rate”, whereas on the previous page they write that Nr is “the sum of NH4-N, NO2-N and NO3-N”
Response 10
“NO3-N removal rate” is now replaced by “N removal rate”. Line 203 page 6
Point 11
Line 196-199 p 6: information referring the results to Table 2 should be added.
Response 11
The table is the summary of the data presented and reference to this table is now included in the text
Point 12
The presentation of the result should be standardized: the results in Table 2 are presented to the second decimal place, but rounded in the description in the manuscript. For example, line 199 p 6: the number in the description is 5.67 whereas in Table 2 it is 5.7. There is no ± symbol in this place.
Response 12
The number of decimals for the same number throughout the whole manuscript and in the table are corrected accordingly.
Point 13
The Authors do not specify the meaning of “±”- standard deviation?
Response 13
The notation of “±” for standard deviation is now in described in section 2.4 Statistical analysis under the Material and Methods. Page 6, Line 182
Point 14
Line 198 p 6: Pr is 29.5±4.1, whereas the number in Table 2 is 29±4.2
Response 14
The numbers are Corrected now
Point 15
Fig 4 – there is no explanation of dBM
Response 15
dBM is replaced with “X” for dry biomass concentration
Point 16
In the manuscript, different notations of the same units are used, e.g. mg/L or mg L-1
Response 16
mg/L is replaced with mg L-1 throughout the text, tables and figures
Author Response File: 
Author Response.docx
Reviewer 2 Report
In this manuscript, the authors investigated the utilization of microalgae, Chlorella sorokiniana, to treat anaerobically conditioned blackwater for nutrient recovery and effluent quality improvement. The contents presented in the manuscript provide important findings of treating BW, e.g., effect of trace elements and N:P ratio on growth, inhibitive impact of NO2. Overall, the manuscript is very systematic and well written, which deserves publication after addressing minor comments.
Please be consistent with acronyms, e.g., dBM, dBMR.
Line 88 If treated blackwater used was effluent after filtration and UV treatment (Third colum in Table 1), please mention it in the content for clarification.
Line 110 why Ntot and Ptot not reported in Table 1?
Author Response
Point 1
Please be consistent with acronyms, e.g., dBM, dBMR.
Response 1
dBM and dBMR are replaced with “X” for dry biomass concentration in the figures and text
Point 2
Line 88 If treated blackwater used was effluent after filtration and UV treatment (Third column in Table 1), please mention it in the content for clarification.
Response 2
Filtration and UV treatment as post treatment methods is now mentioned in the first paragraph of section 2.1 Line 87.
Point 3
Line 110 why Ntot and Ptot not reported in Table 1?
Response 4
Now included.
Author Response File: Author Response.docx
