APSOIL D 22 01044 - R1 - Reviewer

Applied Soil Ecology
Effect of elevated CO2 on the composition of amoA-type nitrifying and nirS-type

denitrifying bacterial communities in soybean rhizosphere in Mollisols
--Manuscript Draft--
Manuscript Number: APSOIL-D-22-01044R1
Article Type: Short Communication
Section/Category: Microorganism-related Submissions
Keywords: nitrification potential; Denitrification potential; abundance; nitrogen cycling; climate

change; Microbial community
Abstract: Climate change, represented as the increased concentration of atmospheric CO2,

affects the stability of agricultural ecosystems. Soil nitrification and denitrification, the
fundamental processes of N cycling, determine N availability to plants, N2O emissions
and flow of N in ecosystems. However, the responses of specific soil microorganisms
involved in nitrification and denitrification to future elevated CO2 scenarios remain
unknown. We planted four soybean varieties, namely Xiaohuangjin (XHJ), Suinong 14
(SN14), Mufeng 5 (MF5), and Dongsheng 1 (DS1), in Mollisols under ambient CO2
(aCO2, 410 ppm) and elevated CO2 (eCO2, 550 ppm) conditions. Using open top
chambers, we elucidated the responses of amoA-type nitrifier and nirS-type denitrifier
to elevated atmospheric CO2 levels in terms of genes abundance, composition, and
potential activity. We found significant differences in soil nitrification and denitrification
potentials for different CO2 conditions and cultivars (P < 0.05). The interaction between
CO2 concentration and cultivar significantly affected the nitrification potential (P <
0.05). The abundance of amoA and nirS genes was relatively resistant to elevated
CO2. Principal coordinate analysis revealed that elevated CO2 did not shifted the
composition of amoA and nirS communities. The relative abundance dominant
operational taxonomic units (OTUs) in amoA and nirS communities were mainly
affected by the cultivars and elevated CO2, respectively. Spearman correlation showed
that soil pH and water content were the main factors affecting the dominant OTUs in
amoA-type nitrification and nirS-type denitrification communities. Our findings
evaluated the composition of amoA-type nitrifying and nirS-type denitrifying bacterial
communities and their roles in N cycling processes under future elevated CO2
condition, highlighting the importance of cultivars as a driver impacting soil N cycling in
managed soybean system.
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Highlights
1 Highlights
2  Elevated CO2 alone did not affect AOB and nirS-type denitrifying bacterial
3 community structures, but soybean cultivars did.
4  The abundance of amoA and nirS genes did not respond to elevated CO2.
5  Nitrification and denitrification potentials were remarkably affected by elevated
6 atmospheric CO2 and soybean cultivars.
7  Water content, pH, AP, TP, and C/N shape the composition of AOB and
8 nirS-type denitrifying bacterial communities.
9
Response to Reviewers
Dear Editor,
We are submitting here our revised manuscript. The revision was based on your
comments and those of three reviewers on our manuscript APSOIL-D-22-01044. Our
itemized responses are attached below, and the changes made are also annotated in the
revised manuscript.
We hope that the revision will be acceptable for publication in Applied soil Ecology.
Yours sincerely,
Zhenhua Yu (on behalf of the co-authors)
************************************************
******
 Reviewer #1: The short communication entitled "Effect of elevated CO2 on the
composition of amoA and nirS communities in soybean rhizosphere in Mollisols"
by Gao and co-authors treated the effect of increasing atmospheric CO2
concentration on the nitrification and denitrification potentialities using four
soybean cultivars in Mollisols. The authors found that increasing CO2
concentration has showed significant differences in the nitrification and
denitrification processes depending on the cultivar. Generally, the manuscript was
well written using clear understandable English and the results were well
presented. However, I have some basic concerns regarding the material and
methods section. For example, the experimental design/description wasn't
complete, the authors did not give enough information on the duration of the
experiment, the number of samples taken, the season and the climatic conditions.
Moreover, the post-sequencing was ambiguous for me, the authors mentioned
gene copies, communities, alpha and beta diversity in the results section, but no
details were given in the M&M section, like which database was used for
taxonomy assignment? Which microbiota was targeted? And, how? Most
importantly, the authors did not justify assigning functional potentialities of such
processes to the sequencing of genes extracted from the soil, because quantifying
genes from DNA does not mean that these genes are active unlike using mRNA
sequencing. Please find below specific comments:
Response/action: Thank you for your positive assessments. We made substantial
changes to the original manuscript and the specific revisions based on your
suggestions were list as follows.
 Title: instead of the abbreviations, you should write "nitrification and

denitrification genes"
Response/action: Thanks, since there are several nitrification and denitrification genes
and we only focus on amoA in nitrification and nirS in denitrification, thus we
follow your suggestion and also referred some published papers like “Diversity and
distribution of amoA-type nitrifying and nirS-type denitrifying microbial communities
in the Yangtze River estuary” published on the journal of Biogeosciences, and finally
we changed the title to “Effect of elevated CO 2 on the composition of amoA-type
nitrifying and nirS-type denitrifying microbial communities in soybean
rhizosphere in Mollisols”.
 Lines 28-29: What are these? proteins? hormones? .. please specify that you're
talking about genes
Response/action: Thanks, we added the information of genes.
 Lines 34-35: To be clarified otherwise what you did is not what you're being
concluded! If there are no correlations between such genes and the corresponding
processes.
Response/action: Thanks, since there are no significant correlations between such
genes and the corresponding processes. We deleted the descriptions in the abstract,
and only kept it in the main text.
 Line 38: it would be clearer if you say that interactions between nitirifiers and
denitrifiers got more complex and denser within the network instead of "network"
alone.
Response/action: Thanks, one reviewer suggested us to delete the network analysis to
make the research content more focused, so we deleted the content about network
analysis in the revised manuscript.
 Abstract: a conclusion part is missing stating the usefulness of the experiment

conducted.
Response/action: Thanks, we added a summary at the end of the abstract.
 Line 67: "The best understood„" it's better saying the well studied„
Response/action: Thanks, we changed.
 Line 87: what do you mean by "human activities"? do you mean different soil
management strategies?
Response/action: Yes, we mean different soil management strategies, now we use
“different soil management strategies” instead of “human activities” to make it more
specific.
 Line 88: Provide some references, as well as some examples of these contrasting
results
Response/action: Thanks, we provided two references to show the contrasting results.
 Line 121: missing information: Duration of the experiment, season of conducting

the experiment, climate conditions of the Mollisol (annual average temperature
and rainfall...), How many plants in each pot, soil chemistry at the beginning ...
Response/action: Thanks, we added these missing information in the first paragraph
of “Materials and methods” section.
 Line 122: How many samples were collected? did you make a composite
sample? ...
Response/action: Thanks, we collected 24 soil samples in total (two CO2
concentrations × four cultivars × three OTCs replicates). We did not make
composite sample.
 Line 128: Give a brief description, the article needs to be complete by itself...
Response/action: Thanks, we gave the descriptions for nitrification and denitrification
potentials.
 Line 131: Please define those before so the reader would understand what they
are exactly...
Response/action: Thanks, we added more information about nitrification and
denitrification communities, amoA and nirS genes, and why we focused on
amoA-type nitrifying and nirS-type denitrifying bacterial in the introduction
section, all of which can be used as the foreshadowing for the readers could better
understand what they are exactly.
 Line 140: Alpha and beta diversities of what? What was the result of your
sequencing? Which taxonomy have you assigned? Which database have you used
for taxonomy assignment? ...
Response/action: Thanks, we added those above missing information.
 Line 154: No explanation was given of how number of gene copies was
determined!
Response/action: Thanks, we added more detail about the calculation of gene copies
at the end of Materials and methods section.
 Line 246: Following the reasoning of the authors, denitrifiers are the most
affected because plants use Nitrate the most!
Response/action: Yes, that is a good point. Generally speaking, the increased plant N
uptake could threaten the denitrifiers, who also use nitrate as substrate. It is
ambiguous to state the variations of the activities or abundance of nitrifiers and
denitrifiers together. In the revised manuscript, we separated the statements of
decrease or lack of nitrifiers and denitrifiers. Besides AOB, the consumers of NH4+-N
also include anammox microorganisms, AOA and Comammox. Kits et al (2017)
found the isolated comammox strain has the highest substrate affinity of all analyzed
ammonia oxidizer isolates. Thus the content of NH4+-N involves several N-cycling
related processes, their combined responses to elevated CO2 determine the content of
NH4+-N.
 Line 251: How do the authors justify their selection of gene copies/number as a
representative of functionality? because sequencing the soil for such genes
doesn't mean targeting functional genes...
Response/action: Thanks, we realized it is not accurate to use “functional genes” after
you pointed out that. We have a habitual thinking that the functional genes refer to
those genes involved in specific nutrition cycling, different from the general 16S
rRNA gene. To avoid ambiguities, we changed “functional genes” to “genes involved
in nitrification and denitrification”.
 Line 256: You just mentioned the opposite before "induced by the increased plant
N uptake under elevated CO2 conditions"
Response/action: Thanks, in the re-organized discussion, we deleted this statement.
 Line 260: Along the manuscript, the authors are making a big confusion treating
nitrifiers and denitrifiers as if they have the same function! Instead, intrifiers are
using ammonia for energy production and their final released product is still a
form that can be assimilated by the plant (inorganic), however, the denitrifiers are
doing the opposite, they're using the inorganic form for energy releasing the gas
form, which is not assimilated by plants, so competition should be with
denitrifiers not nitrifiers! please clear this confusion along the manuscript.
Response/action: Appreciate for your clarification, after you pointed out, we really
realized the statements among nitrifiers, denitrifiers and plants are not clear! So in the
revised manuscript, we separated the responses of nitrifiers and denitrifiers to elevated
CO2.
 Line 265: How did you conclude so if the denitrification ability was reduced?
Response/action: Thanks, actually denitrification ability was increased, now we
changed.
 Line 270: Another figure representing a correlation analysis between nitrification

and denitrification would be more helpful in discussing such results.
Response/action: Thanks, that’ really a good suggestion. We added this correlation
analysis as shown in Table S7.
 Figure 1 and 2: Please use the same treatment codes as used in the text (i.e., eCO2
and aCO2)
 Reviewer #2: The authors reported on a study determining the effects of elevated
CO2 on soil amoA and nirS communities in a pot experiment planted soybean.
Overall the experiment is well designed, the results are straight forward, are
informative and may be interesting to readers. I think this manuscript needs a
major revision before it can be considered for publication. I have the following
major concerns: I would suggest the authors to condense the abstract since they
gave too much details on their results, but did not highlight the importance of
their study.
Response/action: Thanks, we condense the abstract and added a summary to highlight
the importance of the study.
 For the introduction, I really suggest the authors to highlight the importance of
different plant genotypes on N cycling under CO2 enrichment.
Response/action: Thanks, that is a good suggestion, we added background
information of plant genotypes on N cycling under CO2 enrichment.
 It was not clear what amoA refer to AOA amoA or AOB amoA although from the
sequencing data I learned that it was AOB amoA, right? And please explain why
only nirS-type denitrifiers was determined?
Response/action: Yes, amoA refers to AOB amoA, previously we show this
information at the end of the introduction. To make it more clearly, we added this
information in the abstract part during this revision cycle. We also made supplements
for the details of AOB amoA nitrifiers and nirS denitrifiers to explain why we only
determine these two communities in the introduction part.
 Moreover, a weakness, which was not addressed in the manuscript, is that

molecular analysis was only done at the seed filling state, but not event based.
Response/action: The reason of we focused on seed filling stage was based on two
classical studies of Fehr et al (1971) and Mitchell et al (1971). Fehr et al (1971)
developed stage of development descriptions which can be applied to all soybean
(Glycine max (L.) Merr.) genotypes grown in any environment. In this stage, soybean
plant have the maximum pod size and from this stage, seed size increases until pod
cavity filled at R6. Meanwhile, soybean root growth reach the peak value at this stage.
This stage showed the highest root biomass and ability for nutrient absorption, and
also an important stage for yield formation (Mitchell et al., 1971). Thus, we predicted
soil microbial communities in this stage should have more diversity, abundance or N
absorption, all of which are closely related with the target of this study: microbial
communities involved in N cycling. So we chose this stage as a representative stage to
conduct this study.
Mitchell, R.L. Russell, W.J. Root development and rooting patterns of soybean
(Glycine max (L.) Merrill) evaluated under field conditions1. Agron. J. 1971, 63, 313–
316.
Fehr, W.R., Caviness, C.E., Burmood, D.T., Pennington, J.S. Stage of development
descriptions for soybeans, Glycine Max (L.) Merrill. Crop Science. 1971, 11, 929–
931.
 Line 56: Please give some refs after "all ecosystems"

Response/action: Thanks, we added three refs after "all ecosystems".
 Line 59: Hu et al. 2001, this study was conducted in a grassland or did I get it
wrong. However, Hu et al. was really a good ref for elevated CO2 studies and I
think you it would be better to cite this study after "all ecosystems" in line 56
Response/action: Yes, you are exactly right, Hu et al is a grassland reference, and we
moved it to the last sentence after "all ecosystems".
 Line 61-66: please add some refs

Response/action: Thanks, We added.
 Line 72: nitrification is really a contrast process with denitrification?

Response/action: It is not accurate to use “In contrast”, now we removed it.
 Line 88: please add a justification or reference for these inconsistencies.

Response/action: Thanks, we provided two references to show the contrasting results.
 Line 120: please explain why this 80% level was chosen
Response/action: 80% level of the field holding capacity equals to the water content
of 34.5% (w/w), which was based on our previous studies that soybean plants can
grow well under this water content (Jin et al., Crop & Pasture Science, 2011, 62, 563–
570; Li et al., Frontiers in Plant Science, 2017, 8: 1546; Wang et al., European Journal
of Soil Science. 2019; 70: 1212–1220). To make it clear, we added “34.5% (w/w)”
and one references.
 Line 238-242: These refs are not properly credited.

Response/action: Thanks, we reorganized the discussion and credited the refs.
 Line 243: cell viability refers to?

Response/action: In Wan et al (2018), cell viability is a measure of the proportion of
live, healthy cells within a population.
 Line 311-312: indicate or show? It may be not good to end one sentence with a
result in the Discussion section.
Response/action: Thanks, we removed the sentence.
 Line 305: I did not find two sentences are in contrast.

Response/action: Thanks, we deleted “In contrast”.
 Line 313-316: I do not think their results support this.

Response/action: Thanks, we removed the statements.
 I think some editing for English language is required through the manuscript due
to some mistakes.
Response/action: Thanks, we edited the English language with the help of native
English speakers.
 Reviewer #3: This objective study was to measure the effect of elevated CO2 and
soybean cultivars on the abundance and composition of bacterial community
composition of amoA and nirS genes and potential nitrification and denitrification
under controlled conditions. The study has several limitations including targeting
one denitrifier group and one date. The study is not well presented: it focuses too
much on the effect of CO2 and not enough on the effect of cultivars when both
were evaluated. The introduction is lacking contact information, the results are
not well described and the discussion needs to be completely rewritten.
Major comments:
The study evaluated both the effect of elevated CO2 and soybean cultivars but it
focused too much on the elevated CO2, especially since for several
measurements, the effect of cultivars was stronger that the elevated CO2.
Response/action: Thanks, in the revised manuscript, we added predictions and
possible influences of soybean cultivars on nitrification and denitrification.
 There needs to be a sentence at the end of the abstract to highlight the key i.e. the
overall conclusion of the study. The objective is not well formulated and there are
no hypotheses.
Response/action: Thanks, we rephrased the objective and set up hypotheses in the
revised manuscript.
 There is a lack of context in the introduction on the effect of elevated CO2 and
soybean cultivars on nitrifiers and denitrifiers.
Response/action: Thanks, whereas few studies on the effects of elevated CO2 and
soybean cultivars on nitrifiers and denitrifiers in soybean agrosystem were conducted,
we only added limit context about this topic, such as “Decock and Six (2012) found
no effects of eCO2 on total N2O emissions, but denitrifification derived N2O
emissions were stimulated by eCO2 in soybean agroecosystem”.
 Important details are missing in the material and methods including growth
conditions, information about quantitative PCR and bioinformatic pipeline.
Response/action: Thanks, we added the information of plant growth conditions, the
climate features of the research sites, etc. We also provided more detail information
about qPCR reaction, including the range of DNA concentration used, qPCR solution,
program conditions, R2 values and bioinformatic pipeline or database.
 The plant N uptake was mentioned in the discussion but not presented in the
result section however, it would help interpret the data.
Response/action: Thanks! I agree it is not strong enough to cite the unpublished data.
Actually, we have a dilemma in how to cite this data. The plant N uptake data will be
shown in another paper of my colleague, who focused on the responses of
above-ground plants to elevated CO2, to avoid reuse of data, we finally decided to cite
the data in this study.
 The results of the co-occurrence network analyses are dubious given the low
number of samples. The data is not helpful in the interpretation of the overall
finding of this study and it should be removed.
Response/action: The co-occurrence network analyses have been removed in the
revised manuscript.
 There is a lack of possible explanations for the results observed in the discussion,
some of the statements are speculative or incorrect.
Response/action: Thanks, we realized our explanations are not clear and strong
enough after reviewers pointed out. Now we rewrote the discussion and tried our best
to explain the results more clearly.
 Specific comments
The highlights are unclear:
Highlight 1: Is this the abundance and composition of amoA and nirS genes? Specify
that this co-occurrence networks.
Response/action: Thanks, changed! Because we deleted network related content, we
combined previous Highlight 1 and Highlight 2.
 Highlight 2: is this composition?

Response/action: Thanks, we mean composition, activity or abundance. We added
these detail in the revised manuscript.
 Highlight 3: That there is a significant interaction is not helpful: describing what

changes would be better
Response/action: Yes, I agree with you, because the responses of nitrification and
denitrification potentials to CO2 varied among different cultivars, it is difficult to
describe the significant interactions of CO2 and cultivars with limited words, so we
used the general result (significant or not) to show this results. But we described the
details in the main text.
 Highlight 4: Change OTUs to composition.

 Line 23: What do you mean by environmental protection? I suggest removing it.
Response/action: “environmental protection” we referred to the nitrous oxide, which
is a greenhouse gas, is produced as a byproduct in the processes of nitrification and
denitrification. To be more clearly, we removed the description of environmental
protection and changed it to N2O emissions.
 Line 30: change activity to potential activity.

 Line 35: gene copies abundance
 Line 39: the relative abundance of the dominant OTUs?

Response/action: Yes, we changed.
 Line 62: Remove 'The main cycling processes': denitrification and nitrification
are important to N cycling but there is only part of the cycling, all processes are
important to N cycling.
Response/action: Yes, we agree with you, now we changed the description to “As the
main processes responsible for N2O emission, nitrification and denitrification are ....”
 Line 89-91: This needs to be expanded: what do we know about the effect of
elevated CO2? Also, there needs to be some background information on the effect
of soybean on nitrifiers and denitrifiers.
Response/action: Thanks, we gave some examples about the effect of elevated CO2 on
the N2O emissions and AOB abundance. We also showed background information
about the effect of soybean on nitrifiers and denitrifiers.
 Line 100: Please rephrase. This objective study was to measure the effect of
elevated CO2 and soybean cultivars on the abundance and composition of
bacterial community diversity of amoA and nirS genes and potential nitrification
and denitrification under controlled conditions.
Response/action: Thanks, we rephrased the objective based on your suggestions.
 Line 103-105: This sentence is over stating what the study will achieve and
should be removed.
Response/action: Thanks, we removed.
 Line 108: The growth conditions are not described: what was the photoperiod, the
temperature, etc?
Response/action: We added these information which included the annual mean
maximal and minimal temperatures, annual mean temperature and precipitation,
plants were grown under long-day conditions (16h day/8h light) etc.
 Line 110: what was the rationale to use 550 ppm of CO2?
Response/action: 550 ppm was the CO2 concentration predicted in the year of 2050.
We added this information in the revised manuscript.
 Line 119: The soil texture, total C and N and pH need to be provided.
Response/action: we provided more soil information in the revised manuscript: “The
soil, classified as Mollisol, had a silty clay texture (sand 19%, silt 44% and clay 37%)
and was collected from the tillage layer at a 0–10 cm depth from a farming field
located in Niujia town (45°21′N，126°39′E), Wuchang City, Heilongjiang Province,
China. It had an organic C content of 28.3 g kg-1 soil, total nitrogen of 2.24 g kg-1 soil,
available N of 260 mg kg-1 soil, and a pH of 6.97 (1:5 v/v in H2O). ”
 Line 122: what is the rationale to use the R5 stage?

Response/action: The reason of we focused on R5 stage was based on two classical
studies of Fehr et al (1971) and Mitchell et al (1971). Fehr et al (1971) developed
stage of development descriptions which can be applied to all soybean (Glycine max
(L.) Merr.) genotypes grown in any environment. In R5 stage, soybean plant have the
maximum pod size and from this stage, seed size increases until pod cavity filled at
R6. Meanwhile, soybean root growth reach the peak value at R4-R5 stage. R5 stage
showed the highest root biomass and ability for nutrient absorption, and also an
important stage for yield formation (Mitchell et al., 1971). Thus, we predicted soil
microbial communities in R5 stage should have more diversity, abundance or N
absorption, all of which are closely related with the target of this study: microbial
communities involved in N cycling. So we chose R5 stage as an representative stage
to conduct the study.
Mitchell, R.L. Russell, W.J. Root Development and Rooting Patterns of Soybean
(Glycine max (L.) Merrill) Evaluated Under Field Conditions1. Agron. J. 1971,
63, 313–316.
Fehr, W.R., Caviness, C.E., Burmood, D.T., Pennington, J.S. Stage of development
descriptions for soybeans, Glycine Max (L.) Merrill. Crop Science. 1971, 11,
929–931.
 Line 139: There is a lack of details on the bioinformatic used.

Response/action: we added more details about the bioinformatic used and also the
database or pipeline for data analysis.
 Line 150-151: The master mix used, the PCR reaction (including the range of
DNA concentration used) and program conditions need to be specified. What was
used for a standard curve is required and the descriptors (R2, PCR efficiency,
intercept) are also provided.
Response/action: Thanks, we added more detail information about qPCR reaction,
including the range of DNA concentration used, qPCR solution, program conditions,
R2 values, etc.
 Line 155-160: This is not relevant since there are no differences among
treatments, the averages among treatments for nirS and amoA would be sufficient.
Response/action: Thanks, we deleted redundant information and only kept the average
values of nirS and amoA in different treatments.
 Fig. 1A: the result of the interaction needs to be provided with letters. Also, the
results need to be described (lines 163-165).
Response/action: Thanks, we added LSD value and vertical bars in revised Fig 1 to
show more information of eCO2 × cultivar interaction.
 Line 170-182: this should be in the M&M.

Response/action: Thanks, we agree to move the description “After filtering the quality
of sequences and removing chimeric reads, 8,783 and 9,736 sequences were randomly
selected for further analysis of amoA and nirS communities” to M&M, but kept the
information of OTU number, α-diveristy and the distribution of phylum, because we
think these values were calculated results.
 Line 186-192: Were all cultivars different or not? A PERMANOVA and pairwise
comparisons should be done.
Response/action: We added the PERMANOVA (Adonis) analysis between every two
cultivars in the revised Table S3. The PERMANOVA analysis showed that MF5 had
significant different amoA-type nitrifying bacterial communities with SN14 and
DS1, while for nirS-type denitrifying bacterial communities, besides MF5 had
significant different communities with SN14 and DS1, the variations between XHJ
and SN14, and XHJ and DS1 were also significant.
 Line 193-204: Is this describing the number of sequences per OTUs? Were the
number of sequences per sample normalized? It would not make sense otherwise.
How this was done and analyzed should be included in the M&M. Furthermore,
how CO2 or cultivar affected the abundance should be explained: increased or
decreased?
Response/action: Yes, we mean the number of sequences per OTUs and the
number of sequences per sample was also normalized. We added these
information in the Materials and methods: 8,783 and 9,736 sequences were
randomly selected based on the minimum reads for further analysis of amoA and nirS
communities. The detail variations of OTUs abundance, especially those
dominate OTUs with relative higher abundance were explained.
 Table 1: please specify what data is presented in this table: number of sequences
per OTUs?
Response/action: Yes, values are the number of sequences per OTUs, we added
“Values stand for the average of reads number” in the revised table.
 Line 196-197: please describe how OTU140 response differed among cultivars
and CO2 level.
Response/action: In the rhizosphere of MF5 and SN14, the relative abundance of
OTU 140 increased by 57% and 77%, respectively. While in XHJ, the relative
abundance of OTU 140 was similar under aCO2 and eCO2 condition, and in DS1, the
relative abundance of OTU 140 decreased 62% in eCO2 compared with aCO2.
 Line 259-260: There is no evidence to support this statement, please remove it.
Response/action: We re-wrote the manuscript, and removed this statement.
 Line 262-272: Two issues here: 1) this is the nitrification and denitrification
potentials, so this is not the actual rates of these processes. This needs to be
acknowledged when suggesting that it might affect NO3 concentration and N2O
emissions. 2) it is not the potential nitrification and denitrification of the plant,
but rather of the nitrifiers and denitrifiers in the rhizosphere soil of the plant.
Response/action: Yes, we agree with you. For the first issue, now we emphasis “has
the potential of” and “would indicate the NO3 concentration and N2O emissions in the
field” to show their implications for the actual rates of nitrification and denitrification
processes. For the second issue, we rephrased the sentence to show that we referred
nitrifiers and denitrifiers in the rhizosphere soil of the plant.
 Line 261-272: what do we know about differences in nitrification and

denitrification potentials from the literature? Were the result expected?
Response/action: Thanks, we added the results of nitrification and denitrification
potentials from the literature and compared with the results of this study, which is
acceptable.
 Line 280: composition?

Response/action: Thanks, we refer to community composition, activity or abundance.
Now we added the detail.
 Line 295-297: Please remove 'these results revealed'. This is not what this study is
about.
Response/action: Thanks, removed.
 Line 297-299 Most nitrifiers are autotrophic while denitrifiers are heterotrophic
so the fact that more of the dominant denitrifiers OTUs are affected by: why
mention this? , it does not make and is not relevant, please remove it. I think that
this is about explaining why there is more denitrifier OTU affected by elevated
CO2 compared to denitrifiers OTU compared to nitrifiers is not what one would
expect.
Response/action: Thanks, we added the explaination of why more nitrifiers were
affected by soybean cultivars and more denitrifier OTU affected by elevated CO2. The
reasons might be (i) our previous study found different soybean varieties have
different NO 3 - -N uptake capacities, and the extent of increase of NO 3 - -N uptake
under eCO2 differed among cultivars (Li et al., 2017). As the transformation of NO 2 -
to NO 3 - is a reversible reaction, we speculate that the cultivar-induced difference in
NO 3 - -N might influence the nitrification process, in which amoA OTUs evolve, (ii)
for denitrifiers, net primary production (NPP) could be stimulated under elevated CO2,
suggesting that more plant C in soils are expected to increase the demand for
microbial N and threaten the production of NO 3 - (Pendall et al., 2004). Plants also
uptake more NO 3 - under eCO2, which lower the substrate of denitrifers under eCO2.
The limitation and availability of NO 3 - substrates become dominate factors
influencing the denitrifier OTUs.
 Line 307-308: please remove this statement, there is no supporting data for this, it
is too speculative.
 Line 313-314: This statement is incorrect: several studies have used more than
one or two functional genes. Please remove.
 Line 313-327: this is not relevant to the study, please remove it.
Revised manuscript (with changes marked) Click here to view linked References
1 Effect of elevated CO 2 on the composition of amoA-type
2 nitrifying and nirS-type denitrifying bacterial communities in
3 soybean rhizosphere in Mollisols

4
5 Zhiying Gaoa, Lili Guoa, b, Yansheng Lia, Jian Jina, Ying Xua, Xiaobing Liua, Zhenhua
6 Yua*
7
a
8 State Key Laboratory of Black Soils Conservation and Utilization, Northeast Institute
9 of Geography and Agroecology, Chinese Academy of Sciences, 138 Haping Road,
10 Harbin 150081, China
b
11 Institute of Geographical, Henan Academy of Sciences, 64 Longhai Road,
12 Zhengzhou 450052, China
13
14 *Corresponding author:
15 Zhenhua Yu
19 Telephone: +86 451 86602737
20 Fax: +86 451 8660 3736
21 Email: yuzhenhua@iga.ac.cn
22 Abstract
23 Climate change, represented as the increased concentration of atmospheric CO2,

24 affects the stability of agricultural ecosystems. Soil nitrification and denitrification,
25 the fundamental processes of N cycling, determine N availability to plants, N2O
26 emissions and flow of N in ecosystems. However, the responses of specific soil
27 microorganisms involved in nitrification and denitrification to future elevated CO2
28 scenarios remain unknown. We planted four soybean varieties, namely Xiaohuangjin
29 (XHJ), Suinong 14 (SN14), Mufeng 5 (MF5), and Dongsheng 1 (DS1), in Mollisols
30 under ambient CO2 (aCO2, 410 ppm) and elevated CO2 (eCO2, 550 ppm) conditions.
31 Using open top chambers, we elucidated the responses of amoA-type nitrifier and
32 nirS-type denitrifier to elevated atmospheric CO2 levels in terms of genes abundance,
33 composition, and potential activity. We found significant differences in soil
34 nitrification and denitrification potentials for different CO2 conditions and cultivars (P
35 < 0.05). The interaction between CO2 concentration and cultivar significantly affected
36 the nitrification potential (P < 0.05). The abundance of amoA and nirS genes was
37 relatively resistant to elevated CO2. Principal coordinate analysis revealed that
38 elevated CO2 did not shifted the composition of amoA and nirS communities. The
39 relative abundance dominant operational taxonomic units (OTUs) in amoA and nirS
40 communities were mainly affected by the cultivars and elevated CO2, respectively.
41 Spearman correlation showed that soil pH and water content were the main factors
42 affecting the dominant OTUs in amoA-type nitrification and nirS-type denitrification
43 communities. Our findings evaluated the composition of amoA-type nitrifying and
44 nirS-type denitrifying bacterial communities and their roles in N cycling processes
45 under future elevated CO2 condition. This study highlighted the importance of
46 cultivars as a driver impacting soil N cycling in managed soybean system.
47 Keywords
48 Nitrification potential; Denitrification potential; Abundance; Nitrogen cycling;
49 Climate change; Microbial community
50 1. Introduction
51 The concentration of atmospheric carbon dioxide (CO 2 ) has substantially

52 increased from the pre-industrial level of 280 ppm to the current 410 ppm
53 (https://climate.nasa.gov/vital-signs/carbon-dioxide) and is projected to reach 550
54 ppm by the middle of the century according to multi-model predictions
55 (Kuzyakov et al., 2019; Spady et al., 2018). As one of the top three greenhouse
56 gases responsible for climate change, a further increase in CO 2 is expected to
57 influence the biogeochemical cycles of all ecosystems (Chen et al., 2016; De
58 Graaff et al., 2010; Hu et al., 2001). For instance, numerous studies have
59 demonstrated that climate change, mainly characterised by increased
60 atmospheric CO 2 concentrations, significantly impacts the crop yield and
61 stability of agricultural ecosystems (de Graaff et al., 2006; Kassem et al., 2008;
62 Tao et al., 2014; Waqas et al., 2021).
63 Soil nitrogen cycling, which is primarily driven by microbes, is essential for
64 the functioning of natural and agricultural ecosystems. As the main processes
65 responsible for N 2 O emission, nitrification and denitrification are mediated by
66 specific functional organisms and are essential for the N cycling of the
67 plant-soil-microbe continuum. Nitrification is largely performed by nitrifying
68 microbes that gain energy from the oxidisation of NH 3 to NO 2 - or NO 2 - to
69 NO 3 - (Li et al., 2018; Smith et al., 2010; Zhang et al., 2018). Ecologically, the
70 well studied nitrifying microbes are the ammonia oxidisers: ammonia
71 oxidising archaea (AOA) and ammonia oxidising bacteria (AOB) (Prosser and
72 Nicol, 2008), but AOA was reported less sensitive to increases in CO 2
73 concentration (alone or in combination) (Waqas et al., 2021). Other groups of
74 nitrifier include nitrite oxidising bacteria (NOB) which oxidise NO 2 - to NO 3 - ,
75 and commamox bacteria which can complete both steps in nitrification, taking
76 NH 3 all the way to NO 3 - (Daims et al., 2015). Denitrification is the reduction
77 of nitrate toNO2-, NO, N 2 O, and N 2 by denitrifying microbes, which are widely
78 distributed in versatile phylogenetic groups (Zumft, 1992). The reduction of
79 nitrite (NO2-) to NO distinguishes denitrifiers from other nitrate-respiring bacteria.
80 This reaction is regulated by two widely distributed genes nirS and nirK, while nirS
81 gene is found in about 70% of denitrifying bacteria (Tang et al 2010). N 2 O is a
82 greenhouse gas with a global warming potential 300 times greater than CO 2
83 that also contributes to the depletion of stratospheric ozone (Beeckman et al.,
84 2018; Ravishankara et al., 2009). Thus, nitrifying and denitrifying microbes
85 affect not only the regulation of soil N pools and plant -available nitrogen but
86 also the intensity of greenhouse effects, pollution of waterways and
87 groundwater, and other environmental issues (Carnol et al., 2002). Therefore,
88 the responses of nitrification and denitrification and of soil microbial
89 communities to future scenarios of elevated CO2 concentrations should be evaluated.
90 This information would be useful for the development of specific management
91 plans for N control, to maintain the stability and sustainability of agricultural
92 ecosystems, and to manage greenhouse gas emissions. However, the changes in
93 nitrification and denitrification rates, N2O emissions, and functional microbial
94 communities responses to elevated atmospheric CO2 are inconsistencies in
95 agricultural ecosystem, which may be attributed to different crop type, soil nutrient
96 status, and soil managements (Butterly et al., 2016; Hu et al., 2016; Nguyen et al.,
97 2019). For example, Waqas et al. (2021) and Liu et al (2022) showed that elevated
98 CO2 increased N2O emissions and AOB abundance in a paddy soil and a wheat-grown
99 soil, respectively. However, Liu et al also (2022) observed that the elevated CO2
100 significantly affected the composition and communities of AOB, while Waqas et al.
101 (2021) indicated that AOB community structures were unresponsive to long-term CO2
102 enrichment. Decock and Six (2012) found no effect of eCO2 on the total N2O
103 emission in a short term in a soybean-grown field.
104 As the concentration of CO2 in the soil is far higher than in the atmosphere, the
105 effects of elevated CO2 on soil microbes are indirectly mediated by above-ground
106 plants (Kuzyakov et al., 2019). Considering the various root exudates from different
107 plant genotypes that interact with rhizosphere microbiomes, these biochemical
108 processes are genotypically specific (Kawasaki et al., 2016; Vives-Peris et al., 2018).
109 Soybean (Glycine max) is one of the most important legume crops in the world. More
110 than 600 soybean varieties have been released in the Northeast China by the end of
111 the 20th century (Liu et al., 2008). Besides the aforementioned root exudates,
112 different soybean cultivars also have diverse agronomic and physiological
113 characteristics in relevant to N uptake (Li et al., 2017). Thus, the effect of elevated
114 CO2 on N-cycling processes are of utmost importance to soybean N nutrition uptake,
115 yield and protein formation. However, there is little information on the effect of
116 elevated CO2 on nitrification and denitrification among different soybean cultivars in
117 Mollisol, where are more sensitive to climatic fluctuations than those at low latitudes
118 and coastal regions (IPCC, 2013). We hypothesized that both the abundance and
119 structures of amoA-type nitrifying and nirS-type denitrifying bacterial
120 communities in the rhizosphere of soybean would be shifted by eCO2, but the
121 responses would differ between cultivars. To test these hypotheses, with four soybean
122 cultivars grown in typical Mollisols we detected the effect of elevated CO2 and
123 soybean cultivars on the abundance, composition of amoA-type nitrifying and
124 nirS-type denitrifying bacterial communities and nitrification and denitrification
125 potentials in rhizosphere.
126
127 2. Materials and methods

128 A pot experiment was conducted in summer at the agronomy farm of the Northeast
129 Institute of Geography and Agroecology, Harbin, China (45°73’N, 126°61’E). The
130 annual mean maximal and minimal temperatures are 39.2℃ and -42.6℃, respectively,
131 with the annual mean temperature of 4.3℃. The annual mean precipitation is 569 mm.
132 The ambient temperature during the growth season was in a range of 12.7–28.6℃
133 (Zhang et al., 2022). Open top chambers (OTC) with three replicates were used to
134 simulate the elevated atmospheric CO2 concentration (eCO2, 550 ppm), which was the
135 predicted CO2 concentration to reach by 2050 (Houghton et al. 2001). Ambient
136 atmospheric CO2 concentration (aCO2, 410 ppm) was used as a control, and each
137 treatment was conducted in triplicate. The soil, classified as Mollisol, had a silty clay
138 texture (sand 19%, silt 44% and clay 37%). The soil was collected from the tillage
139 layer at a 0–10 cm depth from a farming field located in Niujia town (45°21′N，
140 126°39′E), Wuchang City, Heilongjiang Province, China. It had an organic C content
141 of 28.3 g kg-1 soil, total nitrogen of 2.24 g kg-1 soil and a pH of 6.97 (1:5 v/v in H2O).
142 Four soybean varieties, namely Xiaohuangjin (XHJ), Suinong 14 (SN14), Mufeng 5
143 (MF5), and Dongsheng 1 (DS1), which are widely grown in the Mollisol region of
144 Northeast China, were selected for the pot experiments. Each pot contained two
145 soybean plants and grown under long-day conditions (16h day/8h light). An
146 atmospheric CO2 concentration monitoring system (CI-301, CID Inc., USA) was used
147 to simultaneously monitor and regulate the CO2 concentration inside the open top
148 chambers (Yu et al., 2016). The basal nutrients were thoroughly mixed with the soil
149 before sowing, following the methodology of Jin et al. (2010). Soil moisture was
150 maintained at 80 ± 5% of the field holding capacity (34.5% w/w) during the growth of
151 soybeans (Wang et al., 2019).
152 Finally, 24 soil samples were collected in total (two CO2 concentrations × four
153 cultivars × three OTCs replicates). Soil samples that adhered to the roots were
154 collected using the “root-shaking method” at the initial seed filling (R5) stage (Fehr et
155 al., 1971) (65 days after sowing). After sampling, part of the soil was stored in a
156 refrigerator at -80 °C for DNA extraction. Of the remaining soil samples, some were
157 kept in a refrigerator at 4 °C while the rest were air dried for the analysis of soil
158 physicochemical properties according to the methodology of Yu et al. (2016). The
159 nitrification and denitrification potentials were determined using the methods
160 described by Li and Lu (2019) and Zhou et al. (2012), respectively. For nitrification
161 potential, 10 g of fresh soil and 100 mL medium (1.5 mmoL L-1NH4+, 1.0 mmoL
162 L-1PO43-, pH 7.2) were mixed well in a 250 mL conical flask and incubated on a
163 shaker for 24 h (180 r/min). Ten mL of soil suspension were collected at 2 h, 4 h, 16 h,
164 22 h and 24 h, and centrifuged at 8000 g (4℃) for 10 min before the measurement of
165 NO3--N concentration using a continuous flow analyzer (San++, Skalar, Holland). The
166 growth rate of NO3--N content per hour was used to represent the nitrification
167 potential. Denitrification potential was determined by acetylene reduction method.
168 Briefly, 20 g soil samples and 20 mL sterilized water were put into an incubation
169 bottle and 15 mL (V/V) of air was extracted from the bottle, then an equal volume of
170 acetylene was injected. The bottles were shook at 180 rpm for 1 h to mix the gas and
171 soil thoroughly, and then incubated for 24 h in a 25℃ incubator. After the incubation,
172 1 mL of gas was extracted from the flask with a syringe and the N2O concentration
173 was analyzed by a gas chromatography (Shimadzu GC 2010, Shimane, Japan) and
174 finally converted to N2O per kg of dry soil per hour was used to represent the
175 denitrification potential.
176 Soil microbial DNA was extracted from 0.5 g of frozen soil using the Fast DNA
177 SPIN Kit for Soil (Qbiogene Inc., Carlsbad, CA, USA) according to the
178 manufacturer’s instructions. The functional genes amoA and nirS were amplified
179 using the primer pairs amoA-1F (5-GGGGGTTTCTACTGGTGGT-3') and amoA-2R
180 (5- GGGGTCKGSAAAGCCTTCTTC-3') (Rotthauwe et al., 1997), and nirS-cd3aF
181 (5-GTSAACGTSAAGGARACSGG-3) (Michotey et al., 2000) and nirS-R3cd
182 (5-GASTTCGGRTGSGTCTTGA-3) (Throback et al., 2004), respectively. The
183 purified polymerase chain reaction (PCR) products were normalised to equimolar
184 amounts, combined into one pooled sample, and then sequenced (2 × 300) using an
185 Illumina MiSeq platform at Majorbio Biotechnology (Shanghai, China). After
186 sequencing was completed, the quality of sequence reads was checked using the
187 Quantitative Insights Into Microbial Ecology (QIIME) pipeline (version 1.17;
188 http://qiime.org/). Any ambiguous bases were excluded from further analysis.
189 Sequences with the same barcode were assigned to the same file, and then the barcode
190 and primer sequences were removed. After filtering the quality of sequences and
191 removing chimeric reads, 8,783 and 9,736 sequences were randomly selected based
192 on the minimum reads for further analysis of amoA and nirS communities. Sequences
193 with similarities of >97% were clustered into one OTU. Phylotypes were identified
194 using the Ribosomal Database Project (RDP) pyrosequencing pipeline (http://
195 pyro.cme.msu.edu/). The MOTHUR version 1.30.1 (Schloss et al., 2009) was used to
196 assess the Alpha diversity of amoA-type nitrifying and nirS-type denitrifying bacterial
197 communities. Principal coordinate analysis (PCoA) was performed using the R
198 software package (R3.6.1, www.r-project.org/). The number of sequences per OTU of
199 amoA-type nitrifying and nirS-type denitrifying bacterial communities was
200 normalized and further analysed for the effect of elevated CO2 and cultivar using a
201 two-way analysis of variance (ANOVA). The raw sequences obtained in this study
202 were deposited in GenBank, under the Sequence Read Archive (SRA) accession
203 numbers PRJNA796747 and PRJNA796761 for amoA and nirS communities,
204 respectively. A LightCycler®480 system (Roche Applied Science, Basel, Switzerland)
205 was used to detect copy numbers of the amoA and nirS genes through the quantitative
206 PCR reaction. The primers for those genes were same as the primers in sequencing.
207 The qPCR solution (20 μL) included 1 μL of 10 μM each primer, 10 μL of SYBR
208 Premix Ex TaqTM (Takara, Dalian, China), 1 μL DNA template (DNA concentration:
209 49.5 ng/uL-67.3ng/uL), and 7 μL of sterilized MilliQ water. The program of the
210 quantitative PCR amplifcation started at 95 °C for 30 s, followed by 35 cycles of
211 95 °C for 5 s, and 60 °C for 30 s. Standard curves were established with plasmids that
212 contained the cloned target genes from genomic DNA. The R2 values of those
213 standard curves ranged from 0.990 to 0.999. With the regression equation, the cycle
214 threshold value (Ct) of a DNA sample was converted to the number of genes using
215 standard concentrations and then the gene copy number was calculated as per gram of
216 dry soil.
217
218 3. Results
219 3.1. Nitrification and denitrification potentials and gene copies of amoA and nirS
220 The amoA and nirS gene copy numbers did not respond to elevated CO2. The
221 average amoA gene copies were 3.12 × 104 and 2.73 × 104 copies g-1 dry soil under
222 aCO2 and eCO2 treatments, respectively. The average copy number of nirS gene was
223 3.59 × 106 for aCO2 in comparison to 3.81 × 106 copies g-1 dry soil for eCO2 (Table
224 S1). However, significant differences were observed in the soil nitrification and
225 denitrification potentials for the various CO2 conditions and cultivars (P < 0.001).
226 Moreover, the interaction of CO2 conditions and cultivars significantly affected the
227 nitrification potential (P < 0.001) (Fig. 1), while did not affected the denitrification
228 potential. Further analysis showed that there was no correlation between amoA and
229 nirS gene copies and the nitrification or denitrification potentials (Table S2).
230
231 3.2. Diversity and structure of amoA and nirS communities

232 For the amoA community, the average numbers of OTUs were 61 (aCO2) and 57
233 (eCO2), the Shannon indexes were 2.88 (aCO2) and 2.83 (eCO2), and the ACE indexes
234 were 62.6 (aCO2) and 58.42 (eCO2) (Table S1). For the nirS community, the average
235 numbers of OTUs were 717 (aCO2) and 738 (eCO2), the Shannon indexes were 4.53
236 (aCO2) and 4.60 (eCO2), and the ACE indexes were 939 (aCO2) and 970 (eCO2). With
237 the exception of the OTU number in the XHJ nirS community, no significant
238 differences were observed among OTU numbers, Shannon indexes, and ACE indexes
239 under aCO2 and eCO2 for both amoA and nirS communities. At the phylum level,
240 most nitrifying and denitrifying microorganisms could not be classified.
241 Proteobacteria was the only phylum that could be classified, with a relative abundance
242 of 60.7–80.5% and 12.3–17.4% in amoA and nirS communities, respectively. At the
243 genus level, the main genera included members of Nitrosospira and Nitrosovibrio and
244 of Rhodanobacter and Azoarcus, which were used to classify the amoA and nirS
245 communities, respectively (Fig. S1).
246 Principal coordinate analysis (PCoA) indicated that elevated CO2 levels did not
247 significantly alter the nitrification and denitrification communities, whereas the
248 cultivars did (Fig. 2). This result was further supported by the analysis of similarities
249 (ANOSIM) [R = 0.15 (P = 0.034) and R = 0.2511 (P = 0.001) for nitrification and
250 denitrification communities, respectively] and the non-parametric multivariate
251 analysis of variance (ADONIS) [R2 = 0.2539 (P < 0.015) and R2 = 0.213 (P = 0.002)
252 for nitrification and denitrification communities, respectively]. The PERMANOVA
253 (Adonis) showed that amoA-type nitrifying bacterial communities in the
254 rhizosphere of MF5 significantly differed from SN14 and DS1. The nirS-type
255 denitrifying bacterial communities in the rhizosphere of MF5 were different
256 from SN14 and DS1. Significant difference was also found between XHJ and SN14,
257 and XHJ and DS1, respectively (Table S3).
258 In the amoA-type nitrifying bacterial communities, only OTU 156 was
259 significantly affected by elevated CO2, with the relative abundance decreased in the
260 cultivars of XHJ and SN14. 7 OTUs were significantly affected by cultivars (P <
261 0.05). Specifically, the relative abundance of OTU116, OTU129, OTU141 increased
262 in DS1, whereas the relative abundance of OTU93, OTU123, OTU156 and OTU170
263 decreased in DS1. The response of OTU 140 to elevated CO2 was dependent on the
264 cultivar (P < 0.05). In the rhizosphere of MF5 and SN14, the relative abundance of
265 OTU 140 increased by 57% and 77%, respectively. While in XHJ, the relative
266 abundance of OTU 140 was similar under aCO2 and eCO2 condition, and in DS1, the
267 relative abundance of OTU 140 decreased 62% in eCO2 compared with aCO2 (Table
268 1). The selected OTUs accounted for 35%–49% of the total sequences. Among these
269 OTUs, OTU93, OTU123, and OTU170 were negatively correlated with the
270 nitrification potential (P < 0.05), whereas OTU 116 was positively correlated with the
271 nitrification potential (Table S4). In the nirS community, 8 OTUs were significantly
272 affected by the elevated CO2 (P < 0.05), with increased the relative abundance for
273 OTUs 157 and and decreased abundance for OTUs 1836 under eCO2 . These two
274 dominate OTUs and other two OTUs were significantly affected by cultivars (P <
275 0.05), and other two OTUs with lower relative abundance responses to CO2 were
276 dependent on cultivars (P < 0.05) (Table 1). However, none of these OTUs were
277 related to the denitrification potential (Table S5).
278
279 3.3. Factors influencing the dominated/key OTUs of amoA and nirS communities
280 The correlation heatmap showed that available P (AP), total P (TP), C/N, NO3--N,
281 pH, and water content were correlated with the dominant OTUs in the amoA
282 community. In particular, OTUs 93, 123, and 170 were negatively correlated with pH
283 and soil water content, whereas OTU 116 was positively correlated with pH and soil
284 water content (P < 0.05). OTUs 140 and 141 were negatively correlated with TP,
285 whereas OTU 129 was positively correlated with AP (P < 0.05) (Fig. 4A3A). In the
286 nirS community, water content was the most important driver of dominant OTUs,
287 followed by pH, AP, TP, and C/N. OTUs 256, 651, and 1950 were positive correlated
288 with soil water content, whereas OTUs 1932 and 2242 showed the opposite trend (Fig.
289 4B3B).
290
291 4. Discussion
292 4.1. Effects of elevated CO2 on amoA and nirS gene copies and on nitrification and
293 denitrification potentials
294 The elevated atmospheric CO2 concentrations influence soil
295 nitrification/denitrification processes through plant-mediated effects on soil properties
296 (Singh et al., 2010). In general, elevated CO2 influences soil microbial processes by
297 affecting the plant carbon input and below-ground nitrogen uptake. More
298 rhizodeposits and root exudates under eCO2 may increase their activity/abundance
299 (Qiu et al., 2019). In contrast to our hypothesis, we found that the abundances of
300 amoA and nirS gene copies did not respond to elevated CO2. In the case of this study,
301 the differences of NH4+-N between eCO2 and aCO2 were not significant (Table S6),
302 indicating that the substrates availability for nitrifiers were not affected by eCO2.
303 Even if the rhizodeposits increased under elevated CO2, other nitrifier community
304 members, such as AOA, AOB, comammox or even fungi may dominate in
305 competing NH4+-N under eCO2, while we only focused on AOB in this study.
306 Likewise, Kits et al (2017) found the isolated comammox strain had the highest
307 substrate affinity of all analyzed ammonia oxidizer isolates, thus the substrate affinity
308 of comammox may threat the substrates of AOB. For denitrifiers, the increase of
309 plant N uptake under elevated CO2 (unpublished data) may lead to a decrease in soil
310 denitrification substrate concentration, which in turn may cause the decrease or lack
311 of responsive effects of the activities or abundance of denitrifiers (Barnard et al., 2004;
312 Horz et al., 2004; Schleper, 2010). Besides, Wan et al (2018) found that elevated CO2
313 also suppresses the growth of denitrifiers and cell viability to some extent. Thus, the
314 results of this study agreed with those studies which attributed the lack of changes in
315 genes involved in nitrification and denitrification under elevated CO2 to minimal or
316 lack of changes in soil N pools, gross N process, or plant N uptake (Decock and Six,
317 2012; Seneca et al., 2020).
318
319 N2O emission has been shown to be highly related to ammonia-oxidizing

320 microorganisms and denitrifiers, while AOB contributes more production compared
321 with other nitrifiers (Wrage et al., 2001; Kozlowski et al., 2016). Meta-analysis
322 showed that eCO2 increased potential nitrification in terrestrial ecosystems (+28%)
323 and substantially stimulates N2O emission in agroecosystems (by 44%), but those
324 results varied due to experimental conditions, agricultural practices, climate
325 characteristics and soil properties (Du et al., 2022; Gineyts and Niboyet, 2023). The
326 mechanisms of enhanced N2O emissions under eCO2 condition usually stem from (i)
327 eCO2-induced plant C substrate availability induced by CO2 for AOB and denitrifiers
328 (Wu et al., 2017), (ii) altering plant N preference to NH4+ over NO3– and thus
329 stimulating soil denitrifiers and their activities (Qiu et al., 2019), and (iii) a higher
330 proportion of N2O-producing rather than N2O consuming (N2 producing) denitrifiers
331 under eCO2 (Regan, et al., 2011). In this study, the nitrification and denitrification
332 potentials of different soybean cultivars showed different patterns. For instance,
333 elevated CO2 conditions decreased the nitrification but increased denitrification
334 potentials of the cultivar XHJ, thereby demonstrating that this cultivar may has the
335 potential of a low nitrification ability (less NO3- would be transformed) but high
336 denitrification ability (more N2O emission and NO3- leaching). Therefore, under
337 future CO2 conditions, if this cultivar is planted, it is expected to demand more N
338 fertiliser, which would not be beneficial to the economy or the environment.
339 Conversely, SN14 presented higher nitrification ability but lower denitrification
340 ability compared to the other three cultivars, which indicates that this cultivar can
341 transform more NO3- for plants and present a lower NO3- loss, which was supported
342 by the significant negative correlation analysis between nitrification and
343 denitrification in SN14. The variations in nitrification and denitrification potentials of
344 organisms in the rhizosphere soil of different soybean cultivars would act as
345 indicators of the NO3 concentration and N2O emissions in the field and should be
346 considered in future soybean breeding programs.
347
348 4.2. Effects of elevated CO2 on the amoA and nirS communities
349 With the advent of functional marker genes involved in nitrification and
350 denitrification pathways, more comprehensive insights into the community structures
351 of nitrifiers and denitrifiers can be processed. This study demonstrated that the amoA
352 and nirS communities were not significantly affected by elevated CO2 alone but were
353 affected by soybean cultivars (Fig. 2). Accordingly, little or no effect on nitrifier and
354 denitrifier community composition, activity or abundance by elevated CO2 have been
355 reported in the soybean rhizosphere and bulk soil, paddy soil or even managed
356 grassland (Pereira et al., 2011; Seneca et al., 2020; Waqas et al., 2021). However, we
357 observed that elevated CO2 and cultivars affected specific OTUs. In amoA
358 communities, OTUs 93, 116, 123, and 170, which were affiliated with genus
359 Nitrosospira and no-rank or unclassified bacteria, were significantly affected by
360 cultivars, and they were closely related with the nitrification potential. Therefore,
361 significant changes in the nitrification potential might be closely related to changes in
362 specific OTUs, whose activities might be affected under elevated CO2 conditions. For
363 nirS communities, OTUs grouped as norank_p_environmental_samples and
364 unclassified_p_Proteobacteria were primarily affected by CO2. However, none of
365 these OTUs were correlated with the denitrification potential. The significant change
366 in denitrification potential caused by CO2 or soybean cultivars might be a
367 combinatory effect of several OTUs or other types of denitrifiers. Therefore, the
368 results indicate that the dominant OTUs in amoA and nirS communities were mainly
369 driven by cultivars and elevated CO2, respectively. The reasons might be (i) our
370 previous study found different soybean varieties have different NO 3 - -N uptake
371 capacities, and the extent of increase of NO 3 - -N uptake under eCO2 differed among
372 cultivars (Li et al., 2017). As the transformation of NO 2 - to NO 3 - is a reversible
373 reaction, we speculate that the cultivar-induced difference in NO 3 - -N might
374 influence the nitrification process, in which amoA OTUs evolve, (ii) for denitrifiers,
375 net primary production (NPP) could be stimulated under elevated CO2, suggesting
376 that more plant C in soils are expected to increase the demand for microbial N and
377 threaten the production of NO 3 - (Pendall et al., 2004). Plants also uptake more
378 NO 3 - under eCO2, which lower the substrate of denitrifers under eCO2. The
379 limitation and availability of NO 3 - substrates become dominate factors
380 influencing the denitrifier OTUs.
381 The correlation analysis of these dominant OTUs with soil properties showed that
382 water content and pH were the top two factors influencing the dominant OTUs in
383 amoA and nirS communities. Several studies have reported that water content
384 substantially affects redox changes, and anoxic microenvironments are prone to
385 simulate the emission of N2O by ammonia-oxidising bacteria (AOB) (Shen et al.,
386 2021). The maximum soil water content of 34% was reported by Seneca et al. (2020),
387 which was considered as an oxic condition. Regarding soil pH, our results are in line
388 with previous findings in which soil pH is considered an important factor for the
389 growth or activities of nitrifiers and denitrifiers (Shen et al., 2021; Shi et al., 2022).
390
391 5. Conclusions
392 The abundance of amoA and nirS genes did not respond to elevated CO2, whereas
393 nitrification and denitrification potentials were remarkably affected by elevated
394 atmospheric CO2 and soybean cultivars. The nitrification potential was particularly
395 affected by the combined effects of CO2 and soybean cultivar. The abundances of
396 amoA and nirS genes did not show correlation with the nitrification and denitrification
397 potentials. The elevated CO2 alone did not significantly affect the diversity of the
398 amoA or nirS communities; however, the soybean cultivars did. The variation in
399 specific OTUs under elevated CO2 or different soybean cultivars may significantly
400 contribute to the changes in nitrification and denitrification potentials. Soil properties,
401 such as pH and water content, were essential in shaping the dominant OTUs in the
402 nitrifier and denitrifier communities of Mollisols.
403
404 Acknowledgements
405 The authors thank Jinyuan Zhang and Zhihuang Xie for assisting with the pots
406 experiments and soil sampling.
407
408 Funding
409 This research was supported by the National Natural Science Foundation of China
410 (42177435, 32172123), Key Program of Natural Science Foundation of Heilongjiang
411 Province of China (ZD2021D001) and Young Innovation Promotion Council of the
412 Chinese Academy of Sciences (2019233). .
413
414 Declaration of competing interests

415 The authors declare that they have no known competing financial interests or
416 personal relationships that could have appeared to influence the work reported in this
417 paper.
418
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649 Figure legends
650 Fig. 1. Nitrification and denitrification potentials in the rhizosphere of different
651 soybean cultivars under ambient CO2 (aCO2) and elevated CO2 (eCO2) conditions.
652 The asterisk and “ns” indicate significant (P < 0.05) and insignificant differences
653 between the CO2 treatments, respectively. XHJ, MF5, SN14, and DS1 represent the
654 soybean cultivars Xiaohuangjin, Mufeng 5, Suinong 14, and Dongsheng 1,
655 respectively.
656 Fig. 2. Principal coordinate analysis (PCoA) of amoA and nirS communities in the
657 rhizosphere of different soybean cultivars under ambient CO2 (aCO2) and elevated
658 CO2 (eCO2) conditions. Plots are coloured by (A) CO2 condition and (B) soybean
659 cultivars.
660 Fig. 43. Spearman correlation heatmap of dominant OTUs of (A) amoA and (B) nirS
661 communities and soil characteristics including available P, available K, total P, total K,
662 total nitrogen, total carbon, total nitrogen/total carbon, NO3−-N, NH4+-N, pH, and soil
663 water content in the rhizosphere of soybean cultivars under ambient and elevated CO2.
664 The colour intensity of each cell indicates the r value of the correlation irrespective of
665 cultivar and CO2 treatment. Blue indicates negative correlation, and orange indicates
666 positive correlation. Asterisks denote significantly correlated genera (*P < 0.05, **P
667 < 0.01, ***P < 0.001).
668
669
670
Revised manuscript (Clean version)
1 Effect of elevated CO 2 on the composition of amoA-type
2 nitrifying and nirS-type denitrifying bacterial communities in
3 soybean rhizosphere in Mollisols

4
5 Zhiying Gaoa, Lili Guoa, b, Yansheng Lia, Jian Jina, Ying Xua, Xiaobing Liua, Zhenhua
6 Yua*
7
a
b
11 Institute of Geographical, Henan Academy of Sciences, 64 Longhai Road,
12 Zhengzhou 450052, China
13
14 *Corresponding author:
15 Zhenhua Yu
19 Telephone: +86 451 86602737
20 Fax: +86 451 8660 3736
21 Email: yuzhenhua@iga.ac.cn
22 Abstract
23 Climate change, represented as the increased concentration of atmospheric CO2,

24 affects the stability of agricultural ecosystems. Soil nitrification and denitrification,
25 the fundamental processes of N cycling, determine N availability to plants, N2O
26 emissions and flow of N in ecosystems. However, the responses of specific soil
27 microorganisms involved in nitrification and denitrification to future elevated CO2
28 scenarios remain unknown. We planted four soybean varieties, namely Xiaohuangjin
29 (XHJ), Suinong 14 (SN14), Mufeng 5 (MF5), and Dongsheng 1 (DS1), in Mollisols
30 under ambient CO2 (aCO2, 410 ppm) and elevated CO2 (eCO2, 550 ppm) conditions.
31 Using open top chambers, we elucidated the responses of amoA-type nitrifier and
32 nirS-type denitrifier to elevated atmospheric CO2 levels in terms of genes abundance,
33 composition, and potential activity. We found significant differences in soil
34 nitrification and denitrification potentials for different CO2 conditions and cultivars (P
35 < 0.05). The interaction between CO2 concentration and cultivar significantly affected
36 the nitrification potential (P < 0.05). The abundance of amoA and nirS genes was
37 relatively resistant to elevated CO2. Principal coordinate analysis revealed that
38 elevated CO2 did not shifted the composition of amoA and nirS communities. The
39 relative abundance dominant operational taxonomic units (OTUs) in amoA and nirS
40 communities were mainly affected by the cultivars and elevated CO2, respectively.
41 Spearman correlation showed that soil pH and water content were the main factors
42 affecting the dominant OTUs in amoA-type nitrification and nirS-type denitrification
43 communities. Our findings evaluated the composition of amoA-type nitrifying and
44 nirS-type denitrifying bacterial communities and their roles in N cycling processes
45 under future elevated CO2 condition. This study highlighted the importance of
46 cultivars as a driver impacting soil N cycling in managed soybean system.
47 Keywords
48 Nitrification potential; Denitrification potential; Abundance; Nitrogen cycling;
49 Climate change; Microbial community
50 1. Introduction
51 The concentration of atmospheric carbon dioxide (CO 2 ) has substantially
52 increased from the pre-industrial level of 280 ppm to the current 410 ppm
53 (https://climate.nasa.gov/vital-signs/carbon-dioxide) and is projected to reach 550
54 ppm by the middle of the century according to multi-model predictions
55 (Kuzyakov et al., 2019; Spady et al., 2018). As one of the top three greenhouse
56 gases responsible for climate change, a further increase in CO 2 is expected to
57 influence the biogeochemical cycles of all ecosystems (Chen et al., 2016; De
58 Graaff et al., 2010; Hu et al., 2001). For instance, numerous studies have
59 demonstrated that climate change, mainly characterised by increased
60 atmospheric CO 2 concentrations, significantly impacts the crop yield and
61 stability of agricultural ecosystems (de Graaff et al., 2006; Kassem et al., 2008;
62 Tao et al., 2014; Waqas et al., 2021).
63 Soil nitrogen cycling, which is primarily driven by microbes, is essential for
64 the functioning of natural and agricultural ecosystems. As the main processes
65 responsible for N 2 O emission, nitrification and denitrification are mediated by
66 specific functional organisms and are essential for the N cycling of the
67 plant-soil-microbe continuum. Nitrification is largely performed by nitrifying
68 microbes that gain energy from the oxidisation of NH 3 to NO 2 - or NO 2 - to
69 NO 3 - (Li et al., 2018; Smith et al., 2010; Zhang et al., 2018). Ecologically, the
70 well studied nitrifying microbes are the ammonia oxidisers: ammonia
71 oxidising archaea (AOA) and ammonia oxidising bacteria (AOB) (Prosser and
72 Nicol, 2008), but AOA was reported less sensitive to increases in CO 2
73 concentration (alone or in combination) (Waqas et al., 2021). Other groups of
74 nitrifier include nitrite oxidising bacteria (NOB) which oxidise NO 2 - to NO 3 - ,
75 and commamox bacteria which can complete both steps in nitrification, taking
76 NH 3 all the way to NO 3 - (Daims et al., 2015). Denitrification is the reduction
77 of nitrate toNO2-, NO, N 2 O, and N 2 by denitrifying microbes, which are widely
78 distributed in versatile phylogenetic groups (Zumft, 1992). The reduction of
79 nitrite (NO2-) to NO distinguishes denitrifiers from other nitrate-respiring bacteria.
80 This reaction is regulated by two widely distributed genes nirS and nirK, while nirS
81 gene is found in about 70% of denitrifying bacteria (Tang et al 2010). N 2 O is a
82 greenhouse gas with a global warming potential 300 times greater than CO 2
83 that also contributes to the depletion of stratospheric ozone (Beeckman et al.,
84 2018; Ravishankara et al., 2009). Thus, nitrifying and denitrifying microbes
85 affect not only the regulation of soil N pools and plant -available nitrogen but
86 also the intensity of greenhouse effects, pollution of waterways and
87 groundwater, and other environmental issues (Carnol et al., 2002). Therefore,
88 the responses of nitrification and denitrification and of soil microbial
89 communities to future scenarios of elevated CO2 concentrations should be evaluated.
90 This information would be useful for the development of specific management
91 plans for N control, to maintain the stability and sustainability of agricultural
92 ecosystems, and to manage greenhouse gas emissions. However, the changes in
93 nitrification and denitrification rates, N2O emissions, and functional microbial
94 communities responses to elevated atmospheric CO2 are inconsistencies in
95 agricultural ecosystem, which may be attributed to different crop type, soil nutrient
96 status, and soil managements (Butterly et al., 2016; Hu et al., 2016; Nguyen et al.,
97 2019). For example, Waqas et al. (2021) and Liu et al (2022) showed that elevated
98 CO2 increased N2O emissions and AOB abundance in a paddy soil and a wheat-grown
99 soil, respectively. However, Liu et al also (2022) observed that the elevated CO2
100 significantly affected the composition and communities of AOB, while Waqas et al.
101 (2021) indicated that AOB community structures were unresponsive to long-term CO2
102 enrichment. Decock and Six (2012) found no effect of eCO2 on the total N2O
103 emission in a short term in a soybean-grown field.
104 As the concentration of CO2 in the soil is far higher than in the atmosphere, the
105 effects of elevated CO2 on soil microbes are indirectly mediated by above-ground
106 plants (Kuzyakov et al., 2019). Considering the various root exudates from different
107 plant genotypes that interact with rhizosphere microbiomes, these biochemical
108 processes are genotypically specific (Kawasaki et al., 2016; Vives-Peris et al., 2018).
109 Soybean (Glycine max) is one of the most important legume crops in the world. More
110 than 600 soybean varieties have been released in the Northeast China by the end of
111 the 20th century (Liu et al., 2008). Besides the aforementioned root exudates,
112 different soybean cultivars also have diverse agronomic and physiological
113 characteristics in relevant to N uptake (Li et al., 2017). Thus, the effect of elevated
114 CO2 on N-cycling processes are of utmost importance to soybean N nutrition uptake,
115 yield and protein formation. However, there is little information on the effect of
116 elevated CO2 on nitrification and denitrification among different soybean cultivars in
117 Mollisol, where are more sensitive to climatic fluctuations than those at low latitudes
118 and coastal regions (IPCC, 2013). We hypothesized that both the abundance and
119 structures of amoA-type nitrifying and nirS-type denitrifying bacterial
120 communities in the rhizosphere of soybean would be shifted by eCO2, but the
121 responses would differ between cultivars. To test these hypotheses, with four soybean
122 cultivars grown in typical Mollisols we detected the effect of elevated CO2 and
123 soybean cultivars on the abundance, composition of amoA-type nitrifying and
124 nirS-type denitrifying bacterial communities and nitrification and denitrification
125 potentials in rhizosphere.
126
127 2. Materials and methods

128 A pot experiment was conducted in summer at the agronomy farm of the Northeast
129 Institute of Geography and Agroecology, Harbin, China (45°73’N, 126°61’E). The
130 annual mean maximal and minimal temperatures are 39.2℃ and -42.6℃, respectively,
131 with the annual mean temperature of 4.3℃. The annual mean precipitation is 569 mm.
132 The ambient temperature during the growth season was in a range of 12.7–28.6℃
133 (Zhang et al., 2022). Open top chambers (OTC) with three replicates were used to
134 simulate the elevated atmospheric CO2 concentration (eCO2, 550 ppm), which was the
135 predicted CO2 concentration to reach by 2050 (Houghton et al. 2001). Ambient
136 atmospheric CO2 concentration (aCO2, 410 ppm) was used as a control, and each
137 treatment was conducted in triplicate. The soil, classified as Mollisol, had a silty clay
138 texture (sand 19%, silt 44% and clay 37%). The soil was collected from the tillage
139 layer at a 0–10 cm depth from a farming field located in Niujia town (45°21′N，
140 126°39′E), Wuchang City, Heilongjiang Province, China. It had an organic C content
141 of 28.3 g kg-1 soil, total nitrogen of 2.24 g kg-1 soil and a pH of 6.97 (1:5 v/v in H2O).
142 Four soybean varieties, namely Xiaohuangjin (XHJ), Suinong 14 (SN14), Mufeng 5
143 (MF5), and Dongsheng 1 (DS1), which are widely grown in the Mollisol region of
144 Northeast China, were selected for the pot experiments. Each pot contained two
145 soybean plants and grown under long-day conditions (16h day/8h light). An
146 atmospheric CO2 concentration monitoring system (CI-301, CID Inc., USA) was used
147 to simultaneously monitor and regulate the CO2 concentration inside the open top
148 chambers (Yu et al., 2016). The basal nutrients were thoroughly mixed with the soil
149 before sowing, following the methodology of Jin et al. (2010). Soil moisture was
150 maintained at 80 ± 5% of the field holding capacity (34.5% w/w) during the growth of
151 soybeans (Wang et al., 2019).
152 Finally, 24 soil samples were collected in total (two CO2 concentrations × four
153 cultivars × three OTCs replicates). Soil samples that adhered to the roots were
154 collected using the “root-shaking method” at the initial seed filling (R5) stage (Fehr et
155 al., 1971) (65 days after sowing). After sampling, part of the soil was stored in a
156 refrigerator at -80°C for DNA extraction. Of the remaining soil samples, some were
157 kept in a refrigerator at 4°C while the rest were air dried for the analysis of soil
158 physicochemical properties according to the methodology of Yu et al. (2016). The
159 nitrification and denitrification potentials were determined using the methods
160 described by Li and Lu (2019) and Zhou et al. (2012), respectively. For nitrification
161 potential, 10 g of fresh soil and 100 mL medium (1.5 mmoL L-1NH4+, 1.0 mmoL
162 L-1PO43-, pH 7.2) were mixed well in a 250 mL conical flask and incubated on a
163 shaker for 24 h (180 r/min). Ten mL of soil suspension were collected at 2 h, 4 h, 16 h,
164 22 h and 24 h, and centrifuged at 8000 g (4℃) for 10 min before the measurement of
165 NO3--N concentration using a continuous flow analyzer (San++, Skalar, Holland). The
166 growth rate of NO3--N content per hour was used to represent the nitrification
167 potential. Denitrification potential was determined by acetylene reduction method.
168 Briefly, 20 g soil samples and 20 mL sterilized water were put into an incubation
169 bottle and 15 mL (V/V) of air was extracted from the bottle, then an equal volume of
170 acetylene was injected. The bottles were shook at 180 rpm for 1 h to mix the gas and
171 soil thoroughly, and then incubated for 24 h in a 25℃ incubator. After the incubation,
172 1 mL of gas was extracted from the flask with a syringe and the N2O concentration
173 was analyzed by a gas chromatography (Shimadzu GC 2010, Shimane, Japan) and
174 finally converted to N2O per kg of dry soil per hour was used to represent the
175 denitrification potential.
176 Soil microbial DNA was extracted from 0.5 g of frozen soil using the Fast DNA
177 SPIN Kit for Soil (Qbiogene Inc., Carlsbad, CA, USA) according to the
178 manufacturer’s instructions. The functional genes amoA and nirS were amplified
179 using the primer pairs amoA-1F (5-GGGGGTTTCTACTGGTGGT-3') and amoA-2R
180 (5- GGGGTCKGSAAAGCCTTCTTC-3') (Rotthauwe et al., 1997), and nirS-cd3aF
181 (5-GTSAACGTSAAGGARACSGG-3) (Michotey et al., 2000) and nirS-R3cd
182 (5-GASTTCGGRTGSGTCTTGA-3) (Throback et al., 2004), respectively. The
183 purified polymerase chain reaction (PCR) products were normalised to equimolar
184 amounts, combined into one pooled sample, and then sequenced (2 × 300) using an
185 Illumina MiSeq platform at Majorbio Biotechnology (Shanghai, China). After
186 sequencing was completed, the quality of sequence reads was checked using the
187 Quantitative Insights Into Microbial Ecology (QIIME) pipeline (version 1.17;
188 http://qiime.org/). Any ambiguous bases were excluded from further analysis.
189 Sequences with the same barcode were assigned to the same file, and then the barcode
190 and primer sequences were removed. After filtering the quality of sequences and
191 removing chimeric reads, 8,783 and 9,736 sequences were randomly selected based
192 on the minimum reads for further analysis of amoA and nirS communities. Sequences
193 with similarities of >97% were clustered into one OTU. Phylotypes were identified
194 using the Ribosomal Database Project (RDP) pyrosequencing pipeline (http://
195 pyro.cme.msu.edu/). The MOTHUR version 1.30.1 (Schloss et al., 2009) was used to
196 assess the Alpha diversity of amoA-type nitrifying and nirS-type denitrifying bacterial
197 communities. Principal coordinate analysis (PCoA) was performed using the R
198 software package (R3.6.1, www.r-project.org/). The number of sequences per OTU of
199 amoA-type nitrifying and nirS-type denitrifying bacterial communities was
200 normalized and further analysed for the effect of elevated CO2 and cultivar using a
201 two-way analysis of variance (ANOVA). The raw sequences obtained in this study
202 were deposited in GenBank, under the Sequence Read Archive (SRA) accession
203 numbers PRJNA796747 and PRJNA796761 for amoA and nirS communities,
204 respectively. A LightCycler®480 system (Roche Applied Science, Basel, Switzerland)
205 was used to detect copy numbers of the amoA and nirS genes through the quantitative
206 PCR reaction. The primers for those genes were same as the primers in sequencing.
207 The qPCR solution (20 μL) included 1 μL of 10 μM each primer, 10 μL of SYBR
208 Premix Ex TaqTM (Takara, Dalian, China), 1 μL DNA template (DNA concentration:
209 49.5 ng/uL-67.3ng/uL), and 7 μL of sterilized MilliQ water. The program of the
210 quantitative PCR amplifcation started at 95°C for 30 s, followed by 35 cycles of 95°C
211 for 5 s, and 60°C for 30 s. Standard curves were established with plasmids that
212 contained the cloned target genes from genomic DNA. The R2 values of those
213 standard curves ranged from 0.990 to 0.999. With the regression equation, the cycle
214 threshold value (Ct) of a DNA sample was converted to the number of genes using
215 standard concentrations and then the gene copy number was calculated as per gram of
216 dry soil.
217
218 3. Results
219 3.1. Nitrification and denitrification potentials and gene copies of amoA and nirS
220 The amoA and nirS gene copy numbers did not respond to elevated CO2. The
221 average amoA gene copies were 3.12 × 104 and 2.73 × 104 copies g-1 dry soil under
222 aCO2 and eCO2 treatments, respectively. The average copy number of nirS gene was
223 3.59 × 106 for aCO2 in comparison to 3.81 × 106 copies g-1 dry soil for eCO2 (Table
224 S1). However, significant differences were observed in the soil nitrification and
225 denitrification potentials for the various CO2 conditions and cultivars (P < 0.001).
226 Moreover, the interaction of CO2 conditions and cultivars significantly affected the
227 nitrification potential (P < 0.001) (Fig. 1), while did not affected the denitrification
228 potential. Further analysis showed that there was no correlation between amoA and
229 nirS gene copies and the nitrification or denitrification potentials (Table S2).
230
231 3.2. Diversity and structure of amoA and nirS communities

232 For the amoA community, the average numbers of OTUs were 61 (aCO2) and 57
233 (eCO2), the Shannon indexes were 2.88 (aCO2) and 2.83 (eCO2), and the ACE indexes
234 were 62.6 (aCO2) and 58.42 (eCO2) (Table S1). For the nirS community, the average
235 numbers of OTUs were 717 (aCO2) and 738 (eCO2), the Shannon indexes were 4.53
236 (aCO2) and 4.60 (eCO2), and the ACE indexes were 939 (aCO2) and 970 (eCO2). With
237 the exception of the OTU number in the XHJ nirS community, no significant
238 differences were observed among OTU numbers, Shannon indexes, and ACE indexes
239 under aCO2 and eCO2 for both amoA and nirS communities. At the phylum level,
240 most nitrifying and denitrifying microorganisms could not be classified.
241 Proteobacteria was the only phylum that could be classified, with a relative abundance
242 of 60.7–80.5% and 12.3–17.4% in amoA and nirS communities, respectively. At the
243 genus level, the main genera included members of Nitrosospira and Nitrosovibrio and
244 of Rhodanobacter and Azoarcus, which were used to classify the amoA and nirS
245 communities, respectively (Fig. S1).
246 Principal coordinate analysis (PCoA) indicated that elevated CO2 levels did not
247 significantly alter the nitrification and denitrification communities, whereas the
248 cultivars did (Fig. 2). This result was further supported by the analysis of similarities
249 (ANOSIM) [R = 0.15 (P = 0.034) and R = 0.2511 (P = 0.001) for nitrification and
250 denitrification communities, respectively] and the non-parametric multivariate
251 analysis of variance (ADONIS) [R2 = 0.2539 (P < 0.015) and R2 = 0.213 (P = 0.002)
252 for nitrification and denitrification communities, respectively]. The PERMANOVA
253 (Adonis) showed that amoA-type nitrifying bacterial communities in the
254 rhizosphere of MF5 significantly differed from SN14 and DS1. The nirS-type
255 denitrifying bacterial communities in the rhizosphere of MF5 were different
256 from SN14 and DS1. Significant difference was also found between XHJ and SN14,
257 and XHJ and DS1, respectively (Table S3).
258 In the amoA-type nitrifying bacterial communities, only OTU 156 was
259 significantly affected by elevated CO2, with the relative abundance decreased in the
260 cultivars of XHJ and SN14. 7 OTUs were significantly affected by cultivars (P <
261 0.05). Specifically, the relative abundance of OTU116, OTU129, OTU141 increased
262 in DS1, whereas the relative abundance of OTU93, OTU123, OTU156 and OTU170
263 decreased in DS1. The response of OTU 140 to elevated CO2 was dependent on the
264 cultivar (P < 0.05). In the rhizosphere of MF5 and SN14, the relative abundance of
265 OTU 140 increased by 57% and 77%, respectively. While in XHJ, the relative
266 abundance of OTU 140 was similar under aCO2 and eCO2 condition, and in DS1, the
267 relative abundance of OTU 140 decreased 62% in eCO2 compared with aCO2 (Table
268 1). The selected OTUs accounted for 35%–49% of the total sequences. Among these
269 OTUs, OTU93, OTU123, and OTU170 were negatively correlated with the
270 nitrification potential (P < 0.05), whereas OTU 116 was positively correlated with the
271 nitrification potential (Table S4). In the nirS community, 8 OTUs were significantly
272 affected by the elevated CO2 (P < 0.05), with increased the relative abundance for
273 OTUs 157 and and decreased abundance for OTUs 1836 under eCO2 . These two
274 dominate OTUs and other two OTUs were significantly affected by cultivars (P <
275 0.05), and other two OTUs with lower relative abundance responses to CO2 were
276 dependent on cultivars (P < 0.05) (Table 1). However, none of these OTUs were
277 related to the denitrification potential (Table S5).
278
279 3.3. Factors influencing the dominated/key OTUs of amoA and nirS communities
280 The correlation heatmap showed that available P (AP), total P (TP), C/N, NO3--N,
281 pH, and water content were correlated with the dominant OTUs in the amoA
282 community. In particular, OTUs 93, 123, and 170 were negatively correlated with pH
283 and soil water content, whereas OTU 116 was positively correlated with pH and soil
284 water content (P < 0.05). OTUs 140 and 141 were negatively correlated with TP,
285 whereas OTU 129 was positively correlated with AP (P < 0.05) (Fig. 3A). In the nirS
286 community, water content was the most important driver of dominant OTUs, followed
287 by pH, AP, TP, and C/N. OTUs 256, 651, and 1950 were positive correlated with soil
288 water content, whereas OTUs 1932 and 2242 showed the opposite trend (Fig. 3B).
289
290 4. Discussion
291 4.1. Effects of elevated CO2 on amoA and nirS gene copies and on nitrification and
292 denitrification potentials
293 The elevated atmospheric CO2 concentrations influence soil
294 nitrification/denitrification processes through plant-mediated effects on soil properties
295 (Singh et al., 2010). In general, elevated CO2 influences soil microbial processes by
296 affecting the plant carbon input and below-ground nitrogen uptake. More
297 rhizodeposits and root exudates under eCO2 may increase their activity/abundance
298 (Qiu et al., 2019). In contrast to our hypothesis, we found that the abundances of
299 amoA and nirS gene copies did not respond to elevated CO2. In the case of this study,
300 the differences of NH4+-N between eCO2 and aCO2 were not significant (Table S6),
301 indicating that the substrates availability for nitrifiers were not affected by eCO2.
302 Even if the rhizodeposits increased under elevated CO2, other nitrifier community
303 members, such as AOA, AOB, comammox or even fungi may dominate in
304 competing NH4+-N under eCO2, while we only focused on AOB in this study.
305 Likewise, Kits et al (2017) found the isolated comammox strain had the highest
306 substrate affinity of all analyzed ammonia oxidizer isolates, thus the substrate affinity
307 of comammox may threat the substrates of AOB. For denitrifiers, the increase of
308 plant N uptake under elevated CO2 (unpublished data) may lead to a decrease in soil
309 denitrification substrate concentration, which in turn may cause the decrease or lack
310 of responsive effects of the activities or abundance of denitrifiers (Barnard et al., 2004;
311 Horz et al., 2004; Schleper, 2010). Besides, Wan et al (2018) found that elevated CO2
312 also suppresses the growth of denitrifiers and cell viability to some extent. Thus, the
313 results of this study agreed with those studies which attributed the lack of changes in
314 genes involved in nitrification and denitrification under elevated CO2 to minimal or
315 lack of changes in soil N pools, gross N process, or plant N uptake (Decock and Six,
316 2012; Seneca et al., 2020).
317 N2O emission has been shown to be highly related to ammonia-oxidizing
318 microorganisms and denitrifiers, while AOB contributes more production compared
319 with other nitrifiers (Wrage et al., 2001; Kozlowski et al., 2016). Meta-analysis
320 showed that eCO2 increased potential nitrification in terrestrial ecosystems (+28%)
321 and substantially stimulates N2O emission in agroecosystems (by 44%), but those
322 results varied due to experimental conditions, agricultural practices, climate
323 characteristics and soil properties (Du et al., 2022; Gineyts and Niboyet, 2023). The
324 mechanisms of enhanced N2O emissions under eCO2 condition usually stem from (i)
325 eCO2-induced plant C substrate availability induced by CO2 for AOB and denitrifiers
326 (Wu et al., 2017), (ii) altering plant N preference to NH4+ over NO3– and thus
327 stimulating soil denitrifiers and their activities (Qiu et al., 2019), and (iii) a higher
328 proportion of N2O-producing rather than N2O consuming (N2 producing) denitrifiers
329 under eCO2 (Regan, et al., 2011). In this study, the nitrification and denitrification
330 potentials of different soybean cultivars showed different patterns. For instance,
331 elevated CO2 conditions decreased the nitrification but increased denitrification
332 potentials of the cultivar XHJ, thereby demonstrating that this cultivar may has the
333 potential of a low nitrification ability (less NO3- would be transformed) but high
334 denitrification ability (more N2O emission and NO3- leaching). Therefore, under
335 future CO2 conditions, if this cultivar is planted, it is expected to demand more N
336 fertiliser, which would not be beneficial to the economy or the environment.
337 Conversely, SN14 presented higher nitrification ability but lower denitrification
338 ability compared to the other three cultivars, which indicates that this cultivar can
339 transform more NO3- for plants and present a lower NO3- loss, which was supported
340 by the significant negative correlation analysis between nitrification and
341 denitrification in SN14. The variations in nitrification and denitrification potentials of
342 organisms in the rhizosphere soil of different soybean cultivars would act as
343 indicators of the NO3 concentration and N2O emissions in the field and should be
344 considered in future soybean breeding programs.
345
346 4.2. Effects of elevated CO2 on the amoA and nirS communities
347 With the advent of functional marker genes involved in nitrification and
348 denitrification pathways, more comprehensive insights into the community structures
349 of nitrifiers and denitrifiers can be processed. This study demonstrated that the amoA
350 and nirS communities were not significantly affected by elevated CO2 alone but were
351 affected by soybean cultivars (Fig. 2). Accordingly, little or no effect on nitrifier and
352 denitrifier community composition, activity or abundance by elevated CO2 have been
353 reported in the soybean rhizosphere and bulk soil, paddy soil or even managed
354 grassland (Pereira et al., 2011; Seneca et al., 2020; Waqas et al., 2021). However, we
355 observed that elevated CO2 and cultivars affected specific OTUs. In amoA
356 communities, OTUs 93, 116, 123, and 170, which were affiliated with genus
357 Nitrosospira and no-rank or unclassified bacteria, were significantly affected by
358 cultivars, and they were closely related with the nitrification potential. Therefore,
359 significant changes in the nitrification potential might be closely related to changes in
360 specific OTUs, whose activities might be affected under elevated CO2 conditions. For
361 nirS communities, OTUs grouped as norank_p_environmental_samples and
362 unclassified_p_Proteobacteria were primarily affected by CO2. However, none of
363 these OTUs were correlated with the denitrification potential. The significant change
364 in denitrification potential caused by CO2 or soybean cultivars might be a
365 combinatory effect of several OTUs or other types of denitrifiers. Therefore, the
366 results indicate that the dominant OTUs in amoA and nirS communities were mainly
367 driven by cultivars and elevated CO2, respectively. The reasons might be (i) our
368 previous study found different soybean varieties have different NO 3 - -N uptake
369 capacities, and the extent of increase of NO 3 - -N uptake under eCO2 differed among
370 cultivars (Li et al., 2017). As the transformation of NO 2 - to NO 3 - is a reversible
371 reaction, we speculate that the cultivar-induced difference in NO 3 - -N might
372 influence the nitrification process, in which amoA OTUs evolve, (ii) for denitrifiers,
373 net primary production (NPP) could be stimulated under elevated CO2, suggesting
374 that more plant C in soils are expected to increase the demand for microbial N and
375 threaten the production of NO 3 - (Pendall et al., 2004). Plants also uptake more
376 NO 3 - under eCO2, which lower the substrate of denitrifers under eCO2. The
377 limitation and availability of NO 3 - substrates become dominate factors
378 influencing the denitrifier OTUs.
379 The correlation analysis of these dominant OTUs with soil properties showed that
380 water content and pH were the top two factors influencing the dominant OTUs in
381 amoA and nirS communities. Several studies have reported that water content
382 substantially affects redox changes, and anoxic microenvironments are prone to
383 simulate the emission of N2O by ammonia-oxidising bacteria (AOB) (Shen et al.,
384 2021). The maximum soil water content of 34% was reported by Seneca et al. (2020),
385 which was considered as an oxic condition. Regarding soil pH, our results are in line
386 with previous findings in which soil pH is considered an important factor for the
387 growth or activities of nitrifiers and denitrifiers (Shen et al., 2021; Shi et al., 2022).
388
389 5. Conclusions
390 The abundance of amoA and nirS genes did not respond to elevated CO2, whereas
391 nitrification and denitrification potentials were remarkably affected by elevated
392 atmospheric CO2 and soybean cultivars. The nitrification potential was particularly
393 affected by the combined effects of CO2 and soybean cultivar. The abundances of
394 amoA and nirS genes did not show correlation with the nitrification and denitrification
395 potentials. The elevated CO2 alone did not significantly affect the diversity of the
396 amoA or nirS communities; however, the soybean cultivars did. The variation in
397 specific OTUs under elevated CO2 or different soybean cultivars may significantly
398 contribute to the changes in nitrification and denitrification potentials. Soil properties,
399 such as pH and water content, were essential in shaping the dominant OTUs in the
400 nitrifier and denitrifier communities of Mollisols.
401
402 Acknowledgements
403 The authors thank Jinyuan Zhang and Zhihuang Xie for assisting with the pots
404 experiments and soil sampling.
405
406 Funding
407 This research was supported by the National Natural Science Foundation of China
408 (42177435, 32172123), Key Program of Natural Science Foundation of Heilongjiang
409 Province of China (ZD2021D001) and Young Innovation Promotion Council of the
410 Chinese Academy of Sciences (2019233). .
411
412 Declaration of competing interests

413 The authors declare that they have no known competing financial interests or
414 personal relationships that could have appeared to influence the work reported in this
415 paper.
416
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647 Figure legends
648 Fig. 1. Nitrification and denitrification potentials in the rhizosphere of different
649 soybean cultivars under ambient CO2 (aCO2) and elevated CO2 (eCO2) conditions.
650 The asterisk and “ns” indicate significant (P < 0.05) and insignificant differences
651 between the CO2 treatments, respectively. XHJ, MF5, SN14, and DS1 represent the
652 soybean cultivars Xiaohuangjin, Mufeng 5, Suinong 14, and Dongsheng 1,
653 respectively.
654 Fig. 2. Principal coordinate analysis (PCoA) of amoA and nirS communities in the
655 rhizosphere of different soybean cultivars under ambient CO2 (aCO2) and elevated
656 CO2 (eCO2) conditions. Plots are coloured by (A) CO2 condition and (B) soybean
657 cultivars.
658 Fig. 3. Spearman correlation heatmap of dominant OTUs of (A) amoA and (B) nirS
659 communities and soil characteristics including available P, available K, total P, total K,
660 total nitrogen, total carbon, total nitrogen/total carbon, NO3−-N, NH4+-N, pH, and soil
661 water content in the rhizosphere of soybean cultivars under ambient and elevated CO2.
662 The colour intensity of each cell indicates the r value of the correlation irrespective of
663 cultivar and CO2 treatment. Blue indicates negative correlation, and orange indicates
664 positive correlation. Asterisks denote significantly correlated genera (*P < 0.05, **P
665 < 0.01, ***P < 0.001).
666
667
668
Figure1 Click here to access/download;Figure;revised Fig1.jpg
Figure2 Click here to access/download;Figure;Fig2-revised.docx
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Figure3 Click here to access/download;Figure;Fig44.docx
(A) (B)
Fig. 3
Table (Editable version) Click here to access/download;Table (Editable version);Table1-revised.doc
1 Table 1
2 CO2, cultivars and their interactive effects on the relative abundance of OTUs of amoA and nirS communities at the genus and OTU levels. P values less than 0.05 are shown in bold. OTUs were
3 selected based on relative abundance higher than 0.5% of the total reads. Values stand for the average of reads number.
Genus OTU XHJ MF5 SN14 DS1 ANOVA*(P-value)
aCO2 eCO2 aCO2 eCO2 aCO2 eCO2 aCO2 eCO2 CO2 Cultivar CO2 × cultivar
amoA Nitrosospira OTU116 105 ± 35 88 ± 23 145 ± 32 101 ± 5 141 ± 41 491 ± 173 189 ± 102 189 ± 24 0.196 0.044* 0.059
OTU129 50 ± 10 62 ± 39 64 ± 18 93 ± 25 87 ± 23 106 ± 33 115 ± 29 184 ± 50 0.162 0.046* 0.801
OTU140 440 ± 42 431 ± 76 228 ± 16 357 ± 69 277 ± 88 491 ± 39 575 ± 92 356 ± 55 0.535 0.076 0.021*
OTU141 737 ± 69 1094 ± 105 703 ± 64 796 ± 61 552 ± 133 715 ± 52 1202 ± 286 1022 ± 85 0.254 0.011* 0.257
norank_p__environmental_samples OTU93 1413 ± 193 1518 ± 307 1455 ± 199 1379 ± 35 1337 ± 195 902 ± 35 848 ± 249 930 ± 98 0.564 0.031* 0.503
OTU123 634 ± 167 594 ± 150 790 ± 149 747 ± 167 518 ± 147 166 ± 13 377 ± 206 320 ± 92 0.255 0.026* 0.661
OTU156 235 ± 16 151 ± 23 276 ± 28 259 ± 69 273 ± 13 106 ± 24 134 ± 40 141 ± 22 0.015* 0.012* 0.081
unclassified_k__norank_d__Bacteria OTU170 251 ± 53 372 ± 86 305 ± 59 336 ± 80 235 ± 38 113 ± 16 162 ± 57 146 ± 65 0.928 0.021* 0.279
nirS norank_p__environmental_samples OTU157 672 ± 73 709 ± 86 753 ± 39 774 ± 66 509 ± 29 867 ± 130 446 ± 41 593 ± 30 0.020* 0.011* 0.098
OTU256 297 ± 14 199 ± 15 232 ± 9 255 ± 31 410 ± 83 291 ± 54 498 ± 57 410 ± 63 0.001** 0.055 0.469
OTU379 40 ± 14 72 ± 11 46 ± 5 35 ± 5 38 ± 10 64 ± 8 60 ± 10 35 ± 3 0.378 0.359 0.013*
OTU651 49 ± 3 40 ± 8 36 ± 3 43 ± 19 67 ± 15 55 ± 6 88 ± 9 72 ± 17 0.011* 0.374 0.752
OTU1836 963 ± 119 758 ± 55 841 ± 47 673 ± 31 513 ± 37 397 ± 52 497 ± 77 548 ± 43 0.000** 0.027* 0.232
OTU1932 55 ± 3 55 ± 3 56 ± 2 69 ± 9 30 ± 4 60 ± 8 40 ± 7 42 ± 2 0.006** 0.011* 0.048*
OTU1950 50 ± 10 32 ± 3 35 ± 6 31 ± 7 67 ± 11 42 ± 6 81 ± 9 59 ± 6 0.001** 0.006** 0.556
OTU2242 192 ± 5 195 ± 13 164 ± 9 159 ± 14 140 ± 29 116 ± 18 153 ± 28 132 ± 21 0.019* 0.402 0.874
unclassified_p__Proteobacteria OTU1173 76 ± 24 77 ± 32 68 ± 12 7545 ± 26 175 ± 66 100 ± 43 293 ± 98 154 ± 69 0.040* 0.194 0.495
4
Declaration of Interest Statement
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5 Declaration of interests
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☒The authors declare that they have no known competing financial interests or personal relationships
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9 that could have appeared to influence the work reported in this paper.
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11 ☐The authors declare the following financial interests/personal relationships which may be considered
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Applied Soil Ecology

Effect of elevated CO2 on the composition of amoA-type nitrifying and nirS-type

Manuscript Number: APSOIL-D-22-01044R1

Article Type: Short Communication

Section/Category: Microorganism-related Submissions

Keywords: nitrification potential; Denitrification potential; abundance; nitrogen cycling; climate

Abstract: Climate change, represented as the increased concentration of atmospheric CO2,

3 community structures, but soybean cultivars did.

5  Nitrification and denitrification potentials were remarkably affected by elevated

6 atmospheric CO2 and soybean cultivars.

8 nirS-type denitrifying bacterial communities.

 Title: instead of the abbreviations, you should write "nitrification and

 Abstract: a conclusion part is missing stating the usefulness of the experiment

 Line 121: missing information: Duration of the experiment, season of conducting

 Line 270: Another figure representing a correlation analysis between nitrification

 Moreover, a weakness, which was not addressed in the manuscript, is that

 Line 56: Please give some refs after "all ecosystems"

 Line 61-66: please add some refs

 Line 72: nitrification is really a contrast process with denitrification?

 Line 88: please add a justification or reference for these inconsistencies.

 Line 238-242: These refs are not properly credited.

 Line 243: cell viability refers to?

 Line 305: I did not find two sentences are in contrast.

 Line 313-316: I do not think their results support this.

 Highlight 2: is this composition?

 Highlight 3: That there is a significant interaction is not helpful: describing what

 Highlight 4: Change OTUs to composition.

 Line 30: change activity to potential activity.

 Line 39: the relative abundance of the dominant OTUs?

 Line 122: what is the rationale to use the R5 stage?

 Line 139: There is a lack of details on the bioinformatic used.

 Line 170-182: this should be in the M&M.

 Line 261-272: what do we know about differences in nitrification and

 Line 280: composition?

1 Effect of elevated CO 2 on the composition of amoA-type

2 nitrifying and nirS-type denitrifying bacterial communities in

3 soybean rhizosphere in Mollisols

23 Climate change, represented as the increased concentration of atmospheric CO2,

51 The concentration of atmospheric carbon dioxide (CO 2 ) has substantially

127 2. Materials and methods

231 3.2. Diversity and structure of amoA and nirS communities

319 N2O emission has been shown to be highly related to ammonia-oxidizing

414 Declaration of competing interests

1 Effect of elevated CO 2 on the composition of amoA-type

2 nitrifying and nirS-type denitrifying bacterial communities in

3 soybean rhizosphere in Mollisols

23 Climate change, represented as the increased concentration of atmospheric CO2,

127 2. Materials and methods

231 3.2. Diversity and structure of amoA and nirS communities

412 Declaration of competing interests

Genus OTU XHJ MF5 SN14 DS1 ANOVA*(P-value)

OTU129 50 ± 10 62 ± 39 64 ± 18 93 ± 25 87 ± 23 106 ± 33 115 ± 29 184 ± 50 0.162 0.046* 0.801

OTU379 40 ± 14 72 ± 11 46 ± 5 35 ± 5 38 ± 10 64 ± 8 60 ± 10 35 ± 3 0.378 0.359 0.013*

OTU651 49 ± 3 40 ± 8 36 ± 3 43 ± 19 67 ± 15 55 ± 6 88 ± 9 72 ± 17 0.011* 0.374 0.752

OTU1932 55 ± 3 55 ± 3 56 ± 2 69 ± 9 30 ± 4 60 ± 8 40 ± 7 42 ± 2 0.006** 0.011* 0.048*

OTU1950 50 ± 10 32 ± 3 35 ± 6 31 ± 7 67 ± 11 42 ± 6 81 ± 9 59 ± 6 0.001** 0.006** 0.556

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OTU1950 50 ± 10 32 ± 3 35 ± 6 31 ± 7 67 ± 11 42 ± 6 81 ± 9 59 ± 6 0.001 0.006 0.556