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Identifying the Mechanisms behind the Positive Feedback Loop between Nitrogen Cycling and Algal Blooms in a Shallow Eutrophic Lake

Water 2021, 13(4), 524; https://doi.org/10.3390/w13040524

by Yu Yao^1,2, Huaji Liu^1,2,*

, Ruiming Han^1,2

, Dujun Li^1,2 and Limin Zhang^3,4

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Water 2021, 13(4), 524; https://doi.org/10.3390/w13040524

Submission received: 25 December 2020 / Revised: 9 February 2021 / Accepted: 13 February 2021 / Published: 18 February 2021

(This article belongs to the Special Issue The Impact of Climate Change and Human Activities on Aquatic Environments)

Round 1

Reviewer 1 Report

Unfortunately, there was basically zero characterization of harmful algal blooms to go along with the measurements of N-cycling in this manuscript therefore this manuscript does not adequately address this question. The authors point to locations of increased cyanobacteria abundances from a single satellite image presented in Figure 1 (and cite a paper in the caption that does not appear in the references section), however, the dates of the sediment sampling are not noted in the methods section so it is not clear if this relates to the time frame in which they conducted their sediment measurements. Even if the sediment measurements and satellite imagery overlap in time, the characterization of HAB biomass is probably unsuitable to support the conclusions of the paper - it is hard to determine without knowing which satellite collected the imagery and thereby the spatial resolution of the satellite pixels. If the imagery is from a satellite like Sentinel 3, the resolution is quite coarse. Unfortunately, the in text citation for the imagery doesn't appear in the references for me to be able to verify. I would have liked to see corresponding in situ measurements of algal biomass and a characterization of the specific HAB taxa to truly characterize the presence and magnitude of a HAB in a comparative manner to the sediment observations.

While the authors did conduct 16S sequencing, they only characterized diversity to the phylum level. Also important to note is the the characterization of microbial diversity presented in this manuscript is suitable to examine relative abundance, not absolute abundances of a given OTU. Estimations of higher or lower biomass of a give taxa are not valid. Without some measurements that provide some specificity as to which HAB taxa are present and their magnitude (e.g. qPCR derived gene counts of the dominant cyanobacteria taxa), you can't differentiate whether the the trends in sediment cycling are due to HABs in particular (e.g. an effect caused by a specific HAB species) or algal blooms in general.

Given the data presented, I do not think this manuscript can claim that HABs specifically cause a positive feedback loop in nitrogen cycling as stated in the title. That being said, a large amount of monitoring and research for HABs does occur on Lake Taihu. If some of this data concurrent to the study period might be accessible to the authors, relation of the N-cycling data to the general occurrence of HABs could be possible.

Author Response

Reviewer 1

Comments and Suggestions for Authors

General comments:

This manuscript seeks to address an interesting environmental question about interactions between the occurrence of harmful algal blooms and nitrogen flux from sediments. The manuscript presented careful characterization of N cycling at six locations in Lake Taihu and attempted to relate the patterns in cycling to the occurrence of HABs. Unfortunately, there was basically zero characterization of harmful algal blooms to go along with the measurements of N-cycling in this manuscript therefore this manuscript does not adequately address this question.

Reply: Thanks for your suggestion. The decomposition of harmful algal blooms could make the immediate significantly varaitons of the physicochemical properties in the vicinity of the sediment- water interface. Additional, the six sampling sites were located at the different area of Taihu, which could represent the different biomass and decayed degree of harmful algal blooms. Consequently, we attempted to establish the connection between harmful algal blooms and N-cycling via the high-resultion technique. Moreover, we have added the description of the harmful algal blooms to reduce the ambiguity. Furthermore,we have changed the descriptions of introduction to provide sufficient background in the revised MS. The adequately described of research methods as well as the results and the conclusions were also increased in the revised MS. We have increased the readability by the polish company. The invoice of the polish company is attached as follow:

The authors point to locations of increased cyanobacteria abundances from a single satellite image presented in Figure 1 (and cite a paper in the caption that does not appear in the references section), however, the dates of the sediment sampling are not noted in the methods section so it is not clear if this relates to the time frame in which they conducted their sediment measurements.

Reply: Thanks for your suggestion. The cited paper was listed in the references of the revised MS as follow:

Miao, S., Li, Y.Y., Wu, Z.M., Lyu, H., Li, Y.M., Bi, S., Xu, J., Lei, S.H., Mu, M.,Wang, Q., 2020. A Semianalytical Algorithm for Mapping Proportion of Cyanobacterial Biomass in Eutrophic Inland Lakes Based on OLCI Data. IEEE Trans. Geosci. Remote Sensing 58(7), 5148-5161. (Line 970-972)

Moreover, the detailed dates of the sediment sampling are noted in the revised SI as follow:

Table S1. Geographic information of the sampling sites.

Core	the north of Taihu Lake		the east of Taihu Lake				the west of Taihu Lake
	YL	FDG		WXG	DGH-1	DGH-2		XKW
Latitude Longitude	N 31°23′12″	N 31°24′44″		N 31°15′10″	N 31°11′13″	N 31°9′41″		N 31°12′59″
	E 120°7′45″	E 120°0′59″		E 119°58′40″	E 119°57′15″	E 120°4′4″		E 120°27′39″
Sampling time	Jun 2019	Jul 2019		Jun 2019	Jun 2019	Jun 2019		Jul 2019

The sediment measurements and satellite imagery overlap in time, the characterization of HAB biomass is probably unsuitable to support the conclusions of the paper - it is hard to determine without knowing which satellite collected the imagery and thereby the spatial resolution of the satellite pixels. If the imagery is from a satellite like Sentinel 3, the resolution is quite coarse. Unfortunately, the in text citation for the imagery doesn't appear in the references for me to be able to verify. I would have liked to see corresponding in situ measurements of algal biomass and a characterization of the specific HAB taxa to truly characterize the presence and magnitude of a HAB in a comparative manner to the sediment observations.

Reply: Thanks for your suggestion. The cited paper was listed in the references of the revised MS as follow:

We attempted to select six different sampling sites of Taihu represented the different biomass of harmful alage blooms. Accordingly, related references were cited to establish intuitive impression of different biomass and decayed degree partition in MS. Moreover, we have added the description of algal biomass to truly characterize the presence and magnitude of HABs in the revised Table 1 as follow:

Table 1. The extent of algal bloom hazard, mean values of physicochemical characteristics of overlying water.

Sample sites	pH	ORP (mV)	DO (µM L^-1)	NH₄⁺-N (mg L^-1)	NO₃^--N (mg L^-1)	NO₂^--N (mg L^-1)	SRP (mg L^-1)	Chl-a (μg L^-1)
XKW	6.95	464.28	252.76	0.20	3.78	1.29	2.63	8.23
WXG	7.73	491.38	245.48	0.18	3.71	1.24	5.63	53.04
DGH-1	6.65	141.01	230.25	0.23	3.83	1.25	1.21	61.63
DGH-2	7.74	140.12	210.56	0.11	3.84	1.26	1.78	38.22
FDG	7.3	219.10	260.58	0.22	2.84	1.26	1.31	64.98
YL	7.91	382.43	244.13	0.16	4.34	1.30	0.99	43.21

Reply: Thanks for your suggestion. We have characterized diversity to the class, order, family and genen level but did not descript in the MS. The detailed variation of diversity of class, order, family and genen level were showed as follow:

We conducted a high-resolution sedimentary study of N transformation and its associated microbial activity in Lake Taihu to assess the accumulation rates of the different N fractions in response to HABs, aiming to understand the mechanisms of N cycling in lacustrine environments. Consequently, downcore nitrification and denitrification processes were measured simultaneously in situ in a region of intensified HABs of shallow lake. The sampling sites located at the east of Taihu which with little biomass of HABs were considered as the control group. Moreover, To differentiate whether the the trends in sediment cycling are due to HABs in particular (e.g. an effect caused by a specific HAB species) or algal blooms in general the further indoor experiment were carried out .

Reply: Thanks for your suggestion. There might existed some ambiguity in the MS. We investigated the impacts of sediment HAB decomposition on the N cycling. The decomposition of different biomasses of HABs did not change the main controlling factor on the different N fractions in the profundal sediment. However, the decomposition of the different HAB biomasses led to significantly different nitrification and denitrification processes in sediment. Low HAB biomasses facilitated the classic process of N cycling, with the balanced interaction between nitrification and denitrifica-tion. However, extreme hypoxia in response to high HABs biomasses significantly limited nitrification, which in turn limited denitrification due to the lack of available substrates. The collapse of HABs significantly inhibited denitrification rates, leading to high N accumulation in the sediment; further increased the risk of DON and NH⁴⁺-N release to the overlying water and promoted the widespread occurrence of HABs. Our findings highlight the mechanisms behind the feedback loop linking N and eutrophication in freshwater ecosystems, which can become irreversible without technological intervention. This process is not a positive feedback loop in nitrogen cycling. However, the greenhouse gas emissions of N₂O might be reduced during this process. Consequently, we thik this is not a bad phemomenon between greenhouse gas emissions and freshwater eutrophication.

Author Response File: Author Response.docx

Reviewer 2 Report

This is an interesting manuscript with significant results so it recommended to be published after a thorough exam of nomenclature and Figure/Table captions and legands.

The English in the manuscript is good but the lack of subscripts for compounds (N2O instead of N₂O) is incorrect and distracting.

There is also an absense recognizing the difference between ammonia and ammonium in the text and figures. The authors apparently do not know that only ammonium ions only reside in aqueous solution---ammonia is a gas. This is also a problem in some figure labeling (e.g. Fig 7) but Fig 8 is correct.

The azis labels of some figures (e.g. Fig 4) are partly missing.

The numbering of figures and tables are strange. Why are some figures shown as S1, S2, S4, S5, S6, S9 and S10? Likewise some tables are listed as S2, S3, S4, and S5 while several of the tables and figures are labeled normally.

line 605 of text is "tWe" and probably should be "We"

Author Response

Review2:

Comments and Suggestions for Authors

The English in the manuscript is good but the lack of subscripts for compounds (N2O instead of N₂O) is incorrect and distracting.

Reply: Thanks for your suggestion. All the subscripts and superscripts have been modified with the correct and suitable presentation in the revised manuscript.

Reply: Thanks for your suggestion. I have changed the “ammonia” to “ammonium” to express the ammonium ions in the revised manuscript.

The azis labels of some figures (e.g. Fig 4) are partly missing.

Reply: Thanks for your suggestion. I have modified the format of the figures to display the complete axis labels in the revise manuscript.

Reply: Thanks for your suggestion. The figures with prefix S would be shown in the Supplementary Information as the supplementary figures. Furthermore, the number of the figures and tables have been checked to ensure the consecutiveness.

line 605 of text is "tWe" and probably should be "We”

Reply: Thanks for your suggestion. We have change the “tWe” to “We”. (Line 605)

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I thank the reviewer for their response to my comments. Their comments were helpful in my re-evaluation of the manuscript.

I think that the results presented in this manuscript are novel and interesting findings and worthy of publication. However, even in the revision, I do not find that the authors have made a convincing linkage specifically between HAB biomass and the shift in nitrification and denitrification processes and feel that the use of the term “HAB biomass” throughout the manuscript is lacking some important nuance.

What the authors have demonstrated is that there is a linkage between higher algal biomass (algae as in photosynthetic biomass measured via chlorophyll-a, but not harmful taxa in particular) and these processes. There are no measurements presented of algal toxins or other factors that indicate the biomass measured via chlorophyll-a is truly a harmful algal bloom. It would be more appropriate to use the term “algal biomass” rather then “HAB biomass” throughout the manuscript since they have not provided sufficient evidence that the shift in different nitrification and denitrification processes has been caused by a specific HAB species with the currently presented results. Nonetheless, I think the study still provides interesting and relevant results about how increased algal biomass (whether harmful taxa or not) can cause the sediments to become a source of nitrogen, fueling more algal growth. Therefore, I believe the authors need to revise their manuscript to reflect this nuance.

Additionally, I have a few specific comments for the authors to address:

Introduction

Lines 72 - 74: The lines summarizing the findings of Chen et al 2015b are an incorrect characterization of that paper. Chen et al 2015b does not study the decomposition of any HAB organisms. This paper looked at the organic matter released from two different aquatic plant species, not HABs. These lines should be revised.

Materials and Methods

A description of how chlorophyll-a concentrations were derived should appear in this section since this data now appears in Table 1.

Line 251: typo, “16S rRNA gene abundance”. Additionally, 16S rRNA gene sequencing does not quantify bacterial abundance, it provides relative proportions of which taxa are present in a sample. It is more correct to say “The taxonomic composition of the bacteria in the sediments were characterized using…”

Lines 269 – 276: Please specify if any steps were taken to normalize sequencing data (see comment below on Discussion).

Discussion

Lines 502 – 504: Was there a normalization/rarefaction step taken with the 16S results? If not, we don’t know if the dominance of Sphingobium is due to differences in gene copy number compared to other taxa. This may be an incorrect interpretation of the sequencing data.

Tables and Figures

Table 1: The chlorophyll-a concentration provided in the reviewer response should also appear here.

Figure S10: It would be more appropriate to label the y-axis as “OTU counts” than “OTU values”.

Author Response

I thank the reviewer for their response to my comments. Their comments were helpful in my re-evaluation of the manuscript. I think that the results presented in this manuscript are novel and interesting findings and worthy of publication. However, even in the revision, I do not find that the authors have made a convincing linkage specifically between HAB biomass and the shift in nitrification and denitrification processes and feel that the use of the term “HAB biomass” throughout the manuscript is lacking some important nuance.

Reply: Thanks for your suggestion. The decomposition of algal blooms could consume the oxygen and release the organic matter in the surface sediment as well as the overlying water. The nitrification process would be inhibited because of the exhausted oxygen. Furthermore, the denitrification would be facilitated because of the anerobic and organic-rich microenvironment. Consequently, we attempt to establish the relation between the physicochemical properties variation which with the algal decomposition process and nitrification and denitrification processes. The references could also demonstrate the convincing linkage specifically between algal biomass and the shift in nitrification and denitrification processes. The related references are listed as follow:

Zhu, L., Shi, W.Q., Van Dam, B., Kong, L.W., Yu, J.H., Qing, B.Q., 2020. Algal Accumulation Decreases Sediment Nitrogen Removal by Uncoupling Nitrification-Denitrification in Shallow Eutrophic Lakes. Environmental Science & Technology, 54(10), 6194-6201.

Jiang, X.Y., Gao, G., Zhang, L., Tang, X.M., Shao, K.Q., Hu, Y., 2020. Denitrification and dissimilatory nitrate reduction to ammonium in freshwater lakes of the Eastern Plain, China: Influences of organic carbon and algal bloom. Science of the Total Environment, 710,11.

Chen, X.F., Yang, L.Y., Xiao, L., Miao, A.J., Xi, B.D., 2011. Nitrogen removal by denitrification during cyanobacterial bloom in Lake Taihu. Journal of Freshwater Ecology, 27:2, 243-258.

What the authors have demonstrated is that there is a linkage between higher algal biomass (algae as in photosynthetic biomass measured via chlorophyll-a, but not harmful taxa in particular) and these processes. There are no measurements presented of algal toxins or other factors that indicate the biomass measured via chlorophyll-a is truly a harmful algal bloom. It would be more appropriate to use the term “algal biomass” rather than “HAB biomass” throughout the manuscript since they have not provided sufficient evidence that the shift in different nitrification and denitrification processes has been caused by a specific HAB species with the currently presented results. Nonetheless, I think the study still provides interesting and relevant results about how increased algal biomass (whether harmful taxa or not) can cause the sediments to become a source of nitrogen, fueling more algal growth. Therefore, I believe the authors need to revise their manuscript to reflect this nuance.

Reply: Thanks for your suggestion. We attempt to illustrate the scale of algal biomass via the concentration of chlorophyll-a rather than the algal toxin. Therefore, it would be more appropriate to use the term “algal biomass” rather than “HAB biomass”. To improve the readability, we have modified the description in the revised manuscript as your suggestion. Thank you again for your efforts and work for this manuscript.

Additionally, I have a few specific comments for the authors to address:

Introduction

Reply: Thanks for your suggestion. The original sentence has been modified as “Moreover, previous investigations have demonstrated that the bacterial community composition and functional varied during algae-derived organic matter degradation (Shi et al., 2017).” to make it more appropriate and accurate for the original intention (Lines 77-79).

The cited paper was listed in the references of the revised MS as follow:

Shi, L.M., Huang, Y.X., Zhang, M., Yu, Y., Lu, Y.P., Kong, F.X., 2017. Bacterial community dynamics and functional variation during the long-term decomposition of cyanobacterial blooms in-vitro. Science of The Total Environment. 598, 77-86.

Materials and Methods

A description of how chlorophyll-a concentrations were derived should appear in this section since this data now appears in Table 1.

Reply: Thanks for your suggestion. The method of the chlorophyll-a measurement was presented in the manuscript (Lines 144-146)

Reply: Thanks for your suggestion. The description of the analysis of microbial community composition and diversity in the sediments was presented in Line 280-287. Therefore, we removed the needless sentence (Line 249-250) to avoid the repetitive description.

Lines 269 – 276: Please specify if any steps were taken to normalize sequencing data (see comment below on Discussion).

Reply: Thanks for your suggestion. The detail of the normalize sequencing data has been showed below on Discussion.

Discussion

Lines 502 – 504: Was there a normalization/rarefaction step taken with the 16S results? If not, we don’t know if the dominance of Sphingobium is due to differences in gene copy number compared to other taxa. This may be an incorrect interpretation of the sequencing data.

Reply: Thanks for your suggestion. The V3-V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 515F and 907R by thermocycler PCR system (GeneAmp 9700, ABI, USA). PCR reactions were performed in triplicate 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The resulted PCR products were extracted from a 2% agarose gel and further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor^TM-ST (Promega, USA) according to the manufacturer’s protocol.

Purified amplicons were pooled in equimolar and paired-end sequenced on an Illumina MiSeq platform (Illumina, San Diego, USA) according to the standard protocols by Majorbio Bio-Pharm Technology Co. Ltd. (Shanghai, China).

Raw fastq files were quality-filtered by Trimmomatic and merged by FLASH with the following criteria: (i) The reads were truncated at any site receiving an average quality score <20 over a 50 bp sliding window. (ii) Sequences whose overlap being longer than 10 bp were merged according to their overlap with mismatch no more than 2 bp. (iii) Sequences of each sample were separated according to barcodes (exactly matching) and Primers (allowing 2 nucleotide mismatching), and reads containing ambiguous bases were removed.

(OTUs) were clustered with 97% similarity cutoff using UCHIME (version 7.0.1090, http://www.drive5.com/uparse/) with a novel ‘greedy’ algorithm that performs chimera filtering and OTU clustering simultaneously. The taxonomy of each 16S rRNA gene sequence was analyzed by RDP Classifier algorithm (http://rdp.cme.msu.edu/) against the Silva (SSU123) 16S rRNA database using confidence threshold of 70%.

In order to reduce the effect of variation in sequencing depth, the OTU sequences were rarefied to an equal number per sample.

Tables and Figures

Table 1: The chlorophyll-a concentration provided in the reviewer response should also appear here.

Reply: Thanks for your suggestion. We add the chlorophyll-a concentration in Table 1 as follow:

Table 1. The extent of algal bloom hazard, mean values of physicochemical characteristics of overlying water.

Sample sites	pH	ORP (mV)	DO (µM L^-1)	NH₄⁺-N (mg L^-1)	NO₃^--N (mg L^-1)	NO₂^--N (mg L^-1)	SRP (mg L^-1)	Chl-a (μg L^-1)
XKW	6.95	464.28	252.76	0.20	3.78	1.29	2.63	8.23
WXG	7.73	491.38	245.48	0.18	3.71	1.24	5.63	53.04
DGH-1	6.65	141.01	230.25	0.23	3.83	1.25	1.21	61.63
DGH-2	7.74	140.12	210.56	0.11	3.84	1.26	1.78	38.22
FDG	7.3	219.10	260.58	0.22	2.84	1.26	1.31	64.98
YL	7.91	382.43	244.13	0.16	4.34	1.30	0.99	43.21

Figure S10: It would be more appropriate to label the y-axis as “OTU counts” than “OTU values”.

Reply: Thanks for your suggestion. The description of the y-axis in Figure S10 has been modified to “OTU counts” as follow:

(Please review the image of Figure S10 in the Word file)

Round 3

Reviewer 1 Report

I would like to congratulate the authors on a very nice manuscript and great work. I have no further concerns about the manuscript and look forward to seeing it published!

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Identifying the Mechanisms behind the Positive Feedback Loop between Nitrogen Cycling and Algal Blooms in a Shallow Eutrophic Lake

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