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Functional and Seasonal Changes in the Structure of Microbiome Inhabiting Bottom Sediments of a Pond Intended for Ecological King Carp Farming


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The main goal of the study was to determine changes in the bacterial structure in bottom sediments occurring over the seasons of the year and to estimate microbial metabolic activity. Bottom sediments were collected four times in the year (spring, summer, autumn, and winter) from 10 different measurement points in Cardinal Pond (Ślesin, NW Poland). The Next-Generation Sequencing (MiSeq Illumina) and Community-Level Physiological Profiling techniques were used for identification of the bacterial diversity structure and bacterial metabolic and functional activities over the four seasons. It was evident that Proteobacteria, Acidobacteria, and Bacteroidetes were the dominant phyla, while representatives of Betaproteobacteria, Gammaproteobacteria, and Deltaproteobacteria predominated at the class level in the bottom sediments. An impact of the season on biodiversity and metabolic activity was revealed with the emphasis that the environmental conditions in summer modified the studied parameters most strongly. Carboxylic and acetic acids and carbohydrates were metabolized most frequently, whereas aerobic respiration I with the use of cytochrome C was the main pathway used by the microbiome of the studied bottom sediments.
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Biology 2022, 11, 913.
Functional and Seasonal Changes in the Structure of
Microbiome Inhabiting Bottom Sediments of a Pond Intended
for Ecological King Carp Farming
Agnieszka Wolińska 1,*, Anna Kruczyńska 1, Jarosław Grządziel 2, Anna Gałązka 2, Anna Marzec-Grządziel 2,
Klaudia Szałaj 1 and Agnieszka Kuźniar 1
1 Department of Biology and Biotechnology of Microorganisms, The John Paul II Catholic University of
Lublin, 1 I Konstantynów Str., 20-708 Lublin, Poland; (A.K.); (K.S.); (A.K.)
2 Department of Agriculture Microbiology, Institute of Soil Science and Plant Cultivation, Czartoryskich 8
Str., 24-100 Puławy, Poland; (J.G.); (A.G.); (A.M.-G.)
* Correspondence:
Simple Summary: Bottom sediments are usually classified as extreme habitats for microorganisms.
They are defined as matter deposited on the bottom of water bodies through the sedimentation
process. The quality of sediments is extremely important for the good environmental status of wa-
ter, because they are an integral part of the surface water environment. Microorganisms living in
sediments are involved in biogeochemical transformations and play a fundamental role in main-
taining water purity, decomposition of organic matter, and primary production. As a rule, studies
on bottom sediments focus on monitoring their chemistry and pollution, while little is known about
the structure of bacterial communities inhabiting this extreme environment. In this study, Next-
Generation Sequencing (NGS) was combined with the Community-Level Physiological Profiling
(CLPP) technique to obtain a holistic picture of bacterial biodiversity in the bottom sediments from
Cardinal Pond intended for ecological king carp farming. It was evident that the bottom sediments
of the studied pond were characterized by a rich microbiota composition, whose structure and ac-
tivity depended on the season, and the most extensive modifications of the biodiversity and func-
tionality of microorganisms were noted in summer.
Abstract: The main goal of the study was to determine changes in the bacterial structure in bottom
sediments occurring over the seasons of the year and to estimate microbial metabolic activity. Bot-
tom sediments were collected four times in the year (spring, summer, autumn, and winter) from 10
different measurement points in Cardinal Pond (Ślesin, NW Poland). The Next-Generation Se-
quencing (MiSeq Illumina) and Community-Level Physiological Profiling techniques were used for
identification of the bacterial diversity structure and bacterial metabolic and functional activities
over the four seasons. It was evident that Proteobacteria, Acidobacteria, and Bacteroidetes were the
dominant phyla, while representatives of Betaproteobacteria, Gammaproteobacteria, and Deltapro-
teobacteria predominated at the class level in the bottom sediments. An impact of the season on
biodiversity and metabolic activity was revealed with the emphasis that the environmental condi-
tions in summer modified the studied parameters most strongly. Carboxylic and acetic acids and
carbohydrates were metabolized most frequently, whereas aerobic respiration I with the use of cy-
tochrome C was the main pathway used by the microbiome of the studied bottom sediments.
Keywords: bottom sediments; biodiversity; CLPP; NGS; microbiome; seasonality
Wolińska, A.;
Kruczyńska, A.; Grządziel, J.;
Gałązka, A.;
Marzec-Grządziel, A.;
Szałaj, K.; Kuźniar, A. Functional
and Seasonal Changes in the
Structure of
Microbiome Inhabiting
Sediments of a Pond
Intended for Ecological King Carp
Biology 2022, 11, 913.
Academic Editor:
Pierre Cornelis
Received: 16 May 2022
13 June 2022
14 June 2022
Publisher’s Note:
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tional affiliations.
opyright: © 2022 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article i
s an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://cre-
Biology 2022, 11, 913 2 of 27
1. Introduction
Aquatic microbiota is a term used to describe the vast abundance and diversity of
microorganisms inhabiting water environments [1]. They are estimated to constitute
about 60% of the total biomass and are considered as the most diverse group of organisms
in the entire biosphere [1,2]. In the aquatic environment itself, e.g., in surface waters in-
cluding the oceans, the total number of prokaryotic cells can reach up to 1029, which indi-
cates their undeniable role in the formation and functioning of aquatic ecosystems [1,3].
Microbes perform a number of important ecosystem services in water. Their greatest
function is the primary production of energy from carbon dioxide (CO2) [1] and support-
ing water ecosystems through their involvement in nutrient cycling, especially the nitro-
gen cycle [4]. Usually, high levels of organic carbon are deposited in marine (aquatic) sed-
iments, from which a portion of methane (CH4) is produced [3,5]. Consequently, the ocean
contributes to approx. 2% of the global atmospheric CH4 budget [5,6]. In such conditions,
usually microbial consortia of anaerobic methanotrophic Archaea and sulfate-reducing
bacteria [7] and/or Chloroflexi and Gammaproteobacteria [8], Proteobacteria, Elusimicro-
bia, and Actinobacteria [2] are identified. Cyanobacteria, Deferribacteres, and Thaumar-
chaeota are also abundant, although the taxonomic richness within these phyla is smaller
[2] than in the phyla mentioned above. In shrimp ponds with different population densi-
ties, bacterial communities have been found to be dominated by Gammaproteobacteria,
Alphaproteobacteria, Flavobacteriia, Bacilli, and Actinobacteria [9].
Bottom sediments are composed of sedimentary material deposited on the bottom of
rivers and water bodies. Fish ponds are rich in dissolved organic matter due to intensive
feeding and fecal waste [10]. Ponds accumulate bottom sediments after their basins are
formed under the influence of the water regime (emptying and filling). These sediments
are formed from biological debris originating from the ponds and their catchments, as
well as soil particles and other non-biological materials that have entered the water body
[10]. Cellulolytic bacteria, numerous anaerobic chemoautotrophs, and anaerobic microbi-
ota typically thrive in bottom sediments [10]. In the Arctic sediments in Baffin Bay, Cramm
et al. [11] noted the presence of putative methane-oxidizing Methyloprofundus, sulfate-re-
ducing Desulfobulbaceae, and sulfide-oxidizing Sulfurovum. Lee et al. [12] identified the
presence of Proteobacteria, followed by Chloroflexi, Bacteroidetes, Acidobacteria, and Fir-
micutes in sediments in the Yellow Sea. In the sediments originating from Laoshan Bay
(China), the bacterial community was dominated (>10%) by the phyla Proteobacteria,
Desulfobacterota, and Acidobacteria [13]. It was also emphasized that the biodiversity of
urban ponds, expressed by species richness, appears to be generally lower than in rural
ponds [14].
However, the knowledge of bacterial inhabitants of bottom sediments (especially in
the aspect of Polish stock ponds) is still limited and needs to be constantly extended. This
was an inspiration for conducting the study presented in this paper, where we monitored
changes in the bacterial structure over the seasons of the year taking into account the fact
that temperature is one of the most important environmental drivers with the greatest
influence on the water microbiome [2,15]. What is more, it was suggested that ponds are
highly diverse but understudied systems that could benefit from eDNA monitoring [15].
Usually, assessment of pond biodiversity is regarded as costly, time-consuming, and de-
pendent on taxonomic expertise [13,16]. Nevertheless, a culture-independent technique
was used in this study to identify the highest possible number of bacteria capable of living
in the bottom sediments of a pond intended for organic king carp culture.
Next-Generation Sequencing (NGS) was combined with the Community-Level Phys-
iological Profiling (CLPP) technique to obtain a holistic picture of bacterial biodiversity in
bottom sediments. We hypothesized that both the diversity of bacteria and their metabolic
activity depend on the season of the year. The main goal of the study, in addition to the
precise identification of the taxonomic structure of bacteria inhabiting bottom sediments,
was to find out in which season the microbiome of farm ponds will be the most active and
diverse both structurally and metabolically.
Biology 2022, 11, 913 3 of 27
2. Materials and Methods
2.1. Description of the Breeding Pond (Cardinal Pond in Ślesin)
Bottom sediments for the study were collected from Cardinal Pond located in Ślesin
(53°09′52″ N 17°42′16″ E, Figure 1). Importantly, the Ślesin fish farm is located in an area
that is protected under the European Natura 2000 network. Due to its proximity to the
Noteć River and the Bydgoszcz Canal, the pond is well supplied with fresh and clean
water, creating ideal conditions for king carp farming.
Figure 1. Location of the sampling area on the map of Poland and a view of Cardinal Pond.
Moreover, Cardinal Pond is characterized by a fertile peat bottom, which is an excel-
lent food base for fish. The studied reservoir has an area of 104 hectares (ha). It was built
between 1934 and 1935, and its water surface area is 145.5 ha.
The tradition of carp farming in the Ślesin area is about 100 years old. Carp mature
in ponds for 3 years, and their production is sustainable. The king carp feed on natural
food that has formed in the pond ecosystem, hence the scientific aspect of the current
study related to the assessment of biodiversity in bottom sediments is extremely im-
portant. In addition, the carp are fed with cereals from local crops. In spring (the begin-
ning of April), the pond is stocked with about 80,000 carp crocs (in total about 20–25 tons;
3–4 carp/kg; one croc weighs about 300 g). Then, the fry are fed in summer and caught in
autumn (November) (about 80 tons of carp are harvested), and the water is drained. Dur-
ing winter, the pond has no water, and bottom sediments are exposed to atmospheric
2.2. Bottom Sediment Sampling
Bottom sediment samples were collected from 10 different measurement points (as
independent samples) placed in such a way as to obtain the most representative material
for the study, taking into account the surface area of the pond (Figure 2).
The samples were collected 4 times in the following terms: spring (27 May 2020),
summer (24 August 2020), autumn (26 October 2020), and winter (2 February 2021).
In the wintertime (DecemberMarch), after the fish are removed, the water is drained
from Cardinal Pond and the sediments are exposed to the weather. Additionally, liming
of the substrate is applied to maintain a pH of approximately 7. Therefore, in February
2021, sediment samples were collected from the natural reservoir.
Samples were collected from the assigned points based on GPS locations (Figure 2).
Sediments were sampled from the surface layer of the tank bottom (approximately 1.2 m
depth) using a vacuum-piston sampler (Royal Eijkelkamp, Giesbeek, The Netherlands).
Approximately 1 L of sediments were collected at a time into labeled plastic boxes. To
maintain the natural microbiological processes occurring in the sediments, the boxes were
immediately sealed. The collected samples were stored at 4 °C until they were transported
to the laboratory.
Cardinal Pond
Biology 2022, 11, 913 4 of 27
Figure 2. Location of the bottom sediment sampling points on Cardinal Pond.
2.3. DNA Extraction and NGS Analysis
DNA was isolated using the DNeasy® PowerLyzer® PowerSoil® kit (Qiagen, Hilden,
Germany) as recommended by the manufacturer’s Quick-Start Protocol (Qiagen, German-
town, MD, USA). Approximately 0.300 g of sediment material was used for each of the 10
sampling points (in the relevant seasons), each in triplicate. Metabarcoding of 16S rRNA
was performed within its V3V4 region [17]. 341f and 785r primers were applied both for
the amplification of the mentioned region and for the preparation of the library [17,18].
The PCR was carried out as described by Wolińska et al. [17] with the use of Q5 Hot Start-
High Fidelity 2X Master Mix (New England Biolabs INC., MA, USA). When a positive
effect of PCR was obtained, triplicate DNA isolates of one sample were pooled, which was
in agreement with the recommendation of Kuźniar et al. [19]. Next-Generation Sequenc-
ing (NGS) was performed by Genomed S.A. on a MiSeq sequencer, paired-end (PE) tech-
nology, 2 × 300 nt, using Illumina kit v2.
2.4. Bioinformatic, Functional, and Statistical Analyses
Bioinformatic analyses of the sequences obtained were performed in R v4.1 using
DADA2 v1.18 software [20]. The DECIPHER package v2.20 [20] based on the GreenGenes
v13_8 reference database [21] was applied for classification of the sequences. The results
are presented as relative abundance expressed as a percent of identified sequences at the
phylum, class, and genus taxonomic levels.
The functional profile of the microbial communities was prepared in Linux using
PICRUSt 2.0 software [22]. Predicted genes were compared with the Kyoto Encyclopedia
of Genes and Genomes (KEGG) database. LEfSe analysis and RDA analysis were per-
formed in R using the microeco package (v0.7.1) [23]. The graph was prepared in R using
the heatmap package (v1.0.12) and the ggplot2 package (v3.3.5).
Statistical analyses were performed using STATISTICA 12.0 software. The significant
differences in the KEGG analysis were compared via multiple t-tests.
All identified sequences are available under accession number PRJNA832534
(GenBank Database, NCBI:
cessed on 27 April 2022).
Biology 2022, 11, 913 5 of 27
2.5. Community-Level Physiological Profiling (CLPP)
The metabolic potential of the microbial communities was determined with the use
of the Biolog EcoPlate containing 31 different carbon sources representing five groups
(amines and amides, amino acids, carbohydrates, carboxylic acids, and polymers) [17].
Shortly, 1 g of bottom sediments was weighed, mixed with 99 mL of sterile 0.9% NaCl,
and vortexed (30 min, 150 rpm, 25 °C). Then, all samples were cooled (30 min, 4 °C), trans-
ferred into each of the wells in the EcoPlate, and incubated in the dark (28 °C, 144h). The
experiment was carried out in three replications for each bottom sediment sample. The
results were read every 24 h on the MicroStation ID system.
All results are expressed as a percentage of utilization of individual compounds di-
vided into particular groups and are also presented as a heat map of carbon utilization
patterns of 31 different substrates located on the Biolog EcoPlates. Since the most intensive
metabolic activity was registered after 120 h, the results obtained at this time are presented
in the figures. Average Well Color Development (AWCD) and ShannonWiener (H) indi-
ces were calculated for all the bottom sediment samples [17].
3. Results
3.1. Sequencing Data Quality and Diversity Indices
A summary of the sequencing data quality obtained in the current experiment is pre-
sented in Table S1 (Supplementary Material). A total of 4,729,922 raw sequences were ob-
tained (1,409,576, 1,401,708, 940,019, and 978,559 for all samples collected in spring, summer,
autumn, and winter, respectively). After the filtering step, 3,489,330 sequences remained for
further analysis, i.e., approximately 26.2% did not meet the assumed criteria, and poor-qual-
ity sequences were removed. After that, denoised F/R quality filtering was performed,
which yielded the remaining 3,157,234 (denoised F) and 3,193,392 (denoised R) sequences
for all the analyzed seasons. The total number of merged forward-reverse reads was
2,358,157, whereas 2,307,875 sequences remained after removing chimeras. Consequently,
the relative number of passed reads oscillated in the range of 36–63% (Table S1).
Based on the NGS results, diversity indices were calculated and summarized in Table
S2. Chao1 and ACE are indices estimating the number of species that include both de-
tected and undetected species based on observed species. The analysis of the values of
both indices showed an average value of 119.8 in spring, a lower value of 88.7 in summer,
and values in the range of 105.7 and 103 in autumn and winter, respectively. Species rich-
ness expressed by the Hindex had a mean level of 3.67, 3.41, 3.48, and 3.54 in the bottom
sediment samples collected in spring, summer, autumn, and winter, respectively (Table
S2). Simpson’s index diversity (1-D) had average values of 0.949 (spring), 0.927 (summer),
0.922 (autumn), and 0.943 (winter). In turn, the comparison of the number of genera de-
tected revealed that the bottom sediments contained on average 124 bacterial genera in
spring, 88 in summer, 105 in autumn, and 103 in winter (Table S2).
3.2. Seasonal Changes in the Microbiome Structure—Phylum and Class Taxonomic Level
Seasonal changes in the phyla and classes of the bacteria present in the selected loca-
tions of the bottom sediments in Cardinal Pond are shown in Figure 3 and Figure 4, re-
spectively, whereas the basic chemical characteristics of the studied bottom sediments,
i.e., acidity (pH), redox potential (Eh), and total carbon (TC) in each season, are presented
in Table S3. The bottom sediment pH ranged from 6.87 to 7.87, showing spatial and sea-
sonal variation. Much of the sediment had a pH in the range of 7.4–7.7, with a minimum
in winter and a maximum in summer or autumn. The Eh value showed a fairly wide range
from 126.43 mV to 449.10 mV (Table S3). The seasonal variation of Eh displayed an oppo-
site trend to that of pH. From spring to autumn, the mean Eh values increased linearly
(from 264 to 317 mV) and reached a maximum of 325 mV in winter. TC oscillated between
1.11 and 62.05% and depended on sediment location and seasonality, with a maximum in
autumn and winter.
Biology 2022, 11, 913 6 of 27
The analysis of the bacterial structure at the phylum level indicated that Proteobac-
teria were dominant in the bottom sediments in spring, accounting for 40.80% to 69.88%
of all identified sequences in Sed_10 and Sed_1, respectively (Figure 3).
Acidobacteria were noted as subdominants during the spring season, with relative
abundance ranging from 5.72% to 38% at Sed_3 and Sed_10. Bacteroidetes (0.336.91%),
Verrucomicrobia (2.405.28%), and Ignavibacteriae (accounting for up to 5.21%, but their
presence was not confirmed at Sed_10) were in the third place in the spring bottom sediment
structure (Figure 3). Equally noticeable was the presence of archaeons, represented by Eu-
ryarchaeota, which accounted for more than 1% of the sequences at most sampling points
and whose relative abundance reached 5.04% and 6.76% in Sed_10 and Sed_7, respectively.
Biology 2022, 11, 913 7 of 27
Figure 3. Seasonal changes in the phyla of bacteria in the selected locations of bottom sediment
sampling in Cardinal Pond (1–10 Sedsediment sampling points; Spspring, Susummer, A
autumn, WWinter).
Summer was characterized by the greatest variation in the bacterial structure in the
bottom sediment compared to the other seasons (Figure 3). A comparable share of two
dominant phyla was noted in most of the bottom sediment samples: Proteobacteria ac-
counted for about 21.970.85% of the sequences, while Acidobacteria accounted for 12.2–
61.9%. Summer was the time of their most abundant occurrence in the bottom sediment
samples. There was also an increase in Firmicutes abundance in summer (0.3113.52%)
compared to spring (0.611.70%). Five measurement points also showed a higher
Campilobacterota abundance than in spring (0.138.76%). A similar trend was confirmed
for Euryarchaeota (2.308.47%). In turn, the relative abundance of Bacteroidetes was re-
duced compared to that in spring, accounting for 0.172.99% in summer, similarly to Ig-
navibacteriae (0.313.84%).
The relative abundance of Proteobacteria in autumn and winter was in a similar range,
accounting for about 22.662.05% and 42.3463.30%, respectively. Similarly, the representa-
tives of Acidobacteria constituted 4.9925.76% in the autumn period and 5.1032.15% in
winter (Figure 3). Greater differences were noted in the relative abundance of Bacteroidetes,
Biology 2022, 11, 913 8 of 27
which were more abundant in winter (0.8317.47%) than in autumn (2.756.35%), and Ver-
rucomicrobia (1.4817.41% and 0.8310.13%, winter and autumn, respectively). The abun-
dance of Euryarchaeota was higher in autumn (2.6313.77%) than in winter (1.947.47%), as
in the case of Campilobacterota (0.1212.33% autumn and 0.041.58% winter)
During autumn and winter, there were also a few exceptions in terms of the abun-
dance of individual bacterial phyla depending on the site of sediment collection for anal-
yses. Thus, in autumn, 11.33% of Firmicutes were recorded in Sed_1, while the abundance
of this phylum in the other sediments was in the range of 0.18–2.29% (Figure 3). Analogi-
cally, the abundance of Actinobacteria in Sed_10 in autumn amounted to 55.97%, whereas
a level of 0.11–1.55% was recorded in the other locations. Additionally, during wintertime,
Firmicutes accounted for 9.51% in Sed_2 but extremely lower levels (0.041.52%) were
recorded in the other sediments.
The differences in the bacterial structure at the class taxonomical level in the bottom
sediments are illustrated in Figure 4. In general, the Beta- and Gammaproteobacteria clas-
ses predominated in the bottom sediments collected in spring, autumn, and winter. The
subdominants at the class level depended on the season in which the sediments were sam-
pled, and thus in spring, the Deltaproteobacteria (6.1810.50%) and Acidobacteria_Gp3
(0.7613.05%) classes were noted as subdominants, while Alphaproteobacteria was pre-
dominantly present in sediment samples 2 (18.91%) and 10 (15.93%).
The autumn sampling revealed subdominance of the Acidobacteria_Gp3 (1.03
14.13%) and Deltaproteobacteria (2.707.71%) classes. Noteworthy, there were two excep-
tions, namely the class Actinobacteria with the relative abundance of 55.94% in sediment
sample 10 and Campylobacteria (12.33%) in Sed_7, which were dominant in the structure
of the bottom sediment microbiome (Figure 4).
The subdominance of the Acidobacteria_Gp3 (0.6511.20%) and Deltaproteobacteria
(4.0213.61%) classes was also confirmed in winter; however, it should be noted that the
significant contribution of the Alphaproteobacteria class was confirmed especially in Sed_2
and Sed_6, where the relative abundance of representatives of this class was 21.15% and
7.65%, respectively. Additionally, incidentally but with high relative abundance in the se-
lected sediments samples, representatives of Flavobacteriia (12.7017.27% in Sed_6 and
Sed_7) and Verrucomicrobiae (5.3712.17% in Sed_3, Sed_8, and Sed_9) were recorded.
The analysis of the microbiome structure in the bottom sediments from Cardinal
Pond at the class level revealed that the summer season significantly modified the bacte-
rial composition, which was dramatically different from that in the other seasons (Figure
4). The dominance of the Betaproteobacteria class (16.4939.14%) was shown only in some
sediments numbered 28. In the other sediment sampling points, there was an increase in
the proportion of Acidobacteria_Gp3 (10.9927.19%) and Deltaproteobacteria (7.02
14.90%), while the proportion of Gammaproteobacteria in comparison with the other sea-
sons was lower (1.7711.17%). The relative abundance of the Acidobacteria_Gp13 (0.16
21.20%) and Acidobacteria_Gp18 (1.21–9.78%) classes also increased during the summer
in relation to the other seasons of the year.
The statistical analysis of samples taken in summer and spring revealed statistically
significant differences in the relative abundance of 18 classes, with 14 classified (Figure
S1). In the analysis of summer vs. autumn and summer vs. winter samples, it was 17 clas-
ses. The same analysis was carried out at the genus level (Figure S2). The highest number
of statistically significant differences was observed between samples taken in summer and
winter (117; 42.545% of all analyzed genera). In the comparison of summer samples and
spring samples, it was 108 genera (38.163% of all analyzed genera) and summer vs. au-
tumn106 genera (38.129% of all analyzed genera).
Regardless of the season, the bottom sediments showed the presence of the class
Methanomicrobia, which reached the level of 1.08–5.51% in spring, 1.955.45% in summer,
2.098.73% in autumn, and 1.696.81% in winter (Figure 4).
Biology 2022, 11, 913 9 of 27
Biology 2022, 11, 913 10 of 27
Figure 4. Seasonal changes in the classes of bacteria in the selected locations of bottom sediment
sampling in Cardinal Pond (1–10 Sedsediment sampling points; Spspring, Susummer, A
autumn, WWinter).
3.3. Seasonal Changes in the Microbiome Structure—Genus Taxonomic Level
Seasonal changes in the identified genera of bacteria present in the selected locations
of bottom sediment sampling in Cardinal Pond are presented as heat maps in Figure 5.
It was evident that both the season and the sampling location modified the bacterial
structure in the analyzed bottom sediment samples. The greatest relative abundance of
bacteria that were classified to the taxonomic level of the genus was recorded in spring,
summer, and winter.
Generally, Thiobacillus dominated in the bottom sediments in the spring period. They
constituted c.a. 18% (Sed_3 and Sed_5), 16.60% (Sed_6 and Sed_9), 13.60% (Sed_4 and
Sed_7), and 10.7112.42% (Sed_8 and Sed_1, respectively). In summer, the relative abun-
dance of Thiobacillus was 16.3622.22% (Sed_8 and Sed_4), 6.06–9.85% (Sed_2 and Sed_3),
and 0.283.45% in the other sampling points (Figure 5). In turn, the Thiobacillus abundance
in winter amounted to c.a. 21% (Sed_4 and Sed_5), 16.50% (Sed_3, Sed_8 and Sed_9), and
9.8412.37% (Sed_1 and Sed_7). During autumn, Thiobacillus constituted c.a. 19% (Sed_6)
and 14% (Sed_2, 3, 7 and 9), whilst the highest abundance in this period was exhibited by
Arthrobacter, which reached the value of 55.73% in sed_10.
In the structure of bacteria identified at the genus level, the representatives of Gp3 bac-
teria were indicated as subdominants in all seasons, accounting for approx. 0.8226.76% in
summer, 1.0413.80% in autumn, and similar levels of 0.5411.88% and 0.4110.96% in
spring and winter, respectively (Figure 5). Additionally noteworthy were the representa-
tives of Gp 13 and Gp 18, which were abundant in the microbiome of bottom sediments as
well. In spring, they accounted for 0.625.92% and 0.945.63%, respectively. In summer,
their relative abundance increased to 1.1321.20% (Gp3) and 1.219.78% (Gp 18). They rep-
resented 0.684.44% and 0.696.99%, respectively, in autumn and 0.2012.39% and 0.60
6.11% in winter. The incidental presence of the genera Aeromonas (13.49% in Sed_5 and
5.05% in Sed_8) and Pseudomonas (12.36% in Sed_5) in autumn is noteworthy as well.
Biology 2022, 11, 913 11 of 27
Figure 5. Heat map illustrating seasonal changes in the genera of bacteria in the selected locations
of bottom sediments in Cardinal Pond (1–10 Sedsediment sampling points; Spspring, Susum-
mer, Aautumn, WWinter).
Biology 2022, 11, 913 12 of 27
The description of the bottom sediment microbiome should also emphasize the pres-
ence of genera described as unclassified (Figure A1; Appendix A). In this group, the high-
est frequency in most of the sampling points in Cardinal Pond was exhibited by unclassi-
fied_0052 and unclassified_0102 regardless of the sampling season. The relative seasonal
abundance of unclassified_052 fluctuated around 11% on average, while the unclassi-
fied_0102 group accounted for 9–10% (Figure A1). The Archaea community in the bottom
sediments was represented by c.a. 2.5–3.0% of unclassified_0001.
3.4. Seasonal Changes in the Functional Activity of Bacteria Inhabiting Bottom Sediments
As demonstrated in Figure 6, the highest microbial activity expressed by the AWCD
values (as high as 1.72) was recorded in summer, followed by winter (c.a. 1.60) and au-
tumn (averagely 1.58), whereas the lowest metabolic activity was noted in the samples
collected in spring (c.a. 1.38).
The calculated ShannonWiener index of functional diversity reached the highest
mean level (H = 3.34) in the sediments collected in summer. Slightly lower levels were rec-
orded in autumn and winter (H = 3.30), and the lowest level was noted in spring (H = 3.16).
The catabolic diversity of the microbial community evaluated by substrate utilization
in the Biolog EcoPlate incubated for 120 h is demonstrated in Figure 7. Similar to the bio-
diversity structure, the metabolic activity was influenced by the season of the year.
It was found that carboxylic and acetic acids were the most preferable source of car-
bon for the bottom sediment microorganisms. The compounds were utilized at an average
level of over 30% in the samples collected during summer, autumn, and winter. In turn,
the utilization of these carbon sources in spring amounted to c.a. 29.21% (Figure 7). The
next most readily utilized compounds were carbohydrates, with an average consumption
of about 29% in the sediments collected in spring and winter and 27–28% in autumn and
summer. Amino acids were the third most preferably metabolized carbon source in spring
(19.63%), while their metabolism in the other seasons was just above 18%. The consump-
tion of polymers by the bottom sediment microbiome was estimated to be 16.52% in au-
tumn, 16% in spring, and approx. 14.9% in summer and winter. Amines and amides were
found to be the least readily used carbon sources, with 6.9% consumption in summer,
6.5% in autumn and winter, and 5.9% in spring (Figure 7).
The data provided by the CLPP technique were also visualized as heat maps of car-
bon utilization patterns of 31 substrates from the Biolog EcoPlates (Figure A2). The results
were visualized as standardized data of absorbance measurement at a wavelength of 590
nm with an assumption that higher values mean higher functional activity. The most in-
tensive processes of carbon utilization in the bottom sediment environment were found
in summer, autumn, and winter in contrast to spring, when the processes were less inten-
sive. For example, beta-Methyl-DGlucose, D-Cellobiose, Glucose-1-Phosphate, alpha-Cy-
clodextrin, and L-Threonine were the most intensively metabolized sources of carbon in
the bottom sediments sampled in summer (Figure A2).
Biology 2022, 11, 913 13 of 27
Figure 6. Average Well Color Development (AWCD) and ShannonWiener (H) levels in 10 bottom
sediment sampling points in the four seasons.
Biology 2022, 11, 913 14 of 27
Figure 7. Total carbon source utilization response (%) in 10 bottom sediments sampled during
spring, summer, autumn, and winter for the different carbon substrate groups.
3.5. Bacterial Functional Annotation
The Kyoto Encyclopedia of Genes and Genomes (KEGG) knowledge base was used
to predict the ecological function of bacterial community genes in the different points in
the pond according to the results of classification of the 16S rRNA sequences. It was found
that, in general, the relative abundance of ecological function genes related to carbon (p =
0.000411) and sulfur (p = 0.0000) was significantly higher than those related to nitrogen
and phosphate, with the exception of spring when the highest relative abundance of eco-
logical function genes related to nitrogen was noted in comparison to all the other seasons
(p = 0.0399; Figure 8). This indicates that both the carbon and sulfur cycles in the surface
sediments of the king carp farming pond may be stronger than the nitrogen and phospho-
rus cycles. What is more, a higher number of metabolic pathways were detected by the
KEGG database in Sed_1–6 collected in spring than in the other seasons, which indicates
that the functional activities of the microorganisms are very high during this period of the
year. As evidenced by our results, aerobic respiration I with the use of cytochrome C is
Biology 2022, 11, 913 15 of 27
one of the main metabolic pathways used by microorganisms colonizing the bottom sed-
iments (Figure 8).
Figure 8. Heat map of the pathways annotated by eight pathway clusters (1–8).
Lesser alterations were observed in fermentation metabolism than in the autotrophic
Eubacteria and Archaea metabolism. The predicted bacterial fermentation metabolism,
for example, denitrification, homolactic fermentation, glycolysis III, and mixed acid fer-
mentation, was detected in the members of the active and total community compositions
in all seasons, but particular intensification of these processes was noted in spring (Figure
8). Similarly, TCA cycle VI (obligate autotrophs) and reductive TCA cycles I, V, and VI
were more frequently observed in the total community compositions in spring than in the
other seasons. We also analyzed the CoQ10 pathway characteristic for prokaryotes and
evidenced its maximum in spring in comparison with the other seasons. In the present
study, we predicted L-arginine biosynthesis IV (archaebacteria) pathways in the total bac-
terial community. In detail, the relative abundance of L-arginine biosynthesis IV (archae-
bacteria) pathways showed the highest abundance in the open water habitats and the low-
est abundance in the pond shoreline (Figure 8).
Biology 2022, 11, 913 16 of 27
4. Discussion
The major goal of this study was to identify (using culture-independent techniques)
the composition of the bacterial microbiome, to determine seasonal changes in the struc-
ture of autochthonous bacteria inhabiting the bottom sediments from Cardinal Pond in
Ślesin, and to estimate their metabolic and functional activities.
Using the NGS technique with the Illumina MiSeq platform, bacterial diversity was
determined at different taxonomic levels, from phyla to genera [24]. Importantly, the state-
of-the-art sequencing technology used allowed us to identify the bacterial microbiome
within a group of viable but non-culturable bacteria, which greatly enhances the cognitive
value of this paper and sheds new light on the bacteria present in the bottom sediments,
which still remain rather relatively unrecognized.
Environmental factors are known to shape the structure and function of microbial
communities [9]; hence, the location of the bottom sediment sites in the study pond and
the time of the year (indirectly temperature) influenced the presence of autochthonous
bacteria, as demonstrated in the current study.
The analysis of the bacterial structure at the phylum level revealed the dominance of
Proteobacteria inhabiting the bottom sediments in a large abundance (15–70%) in all sea-
sons of the year, with prevalence in spring (Figure 3) at all sampling points in Cardinal
Pond (Figure 2). Here, it is worth noting that Proteobacteria are heterotrophic, versatile
opportunists and have been extensively studied not only as both pathogens and beneficial
symbionts of plants and animals, but also as ubiquitous organisms that live freely (au-
tochthonously) in many environments [25]. In addition, they find potential applications
in biotechnology as iron-oxidizing bacteria [26] and have an underestimated potential to
produce bioactive molecules [27]; therefore, their presence in bottom sediment seems to
be desirable. Acidobacteria were the second dominant bacterial phylum after Proteobac-
teria. They were present at every bottom sediment collection point in all seasons (Figure
3), with an outstanding relative abundance characterized by seasonal variation, especially
in summer (9.80–62%). It has been indicated that this phylum includes bacteria that are
ubiquitous and abundantly distributed in most ecosystems [28], thus their abundant pres-
ence in the bottom sediments is not surprising. Acidobacteria is an ecologically important
phylum with a set of genes involved in various metabolic pathways. It plays a dynamic
role in ecological processes, namely regulation of biogeochemical cycles, degradation of
biopolymers, and secretion of exopolysaccharides [28]. The third most abundant bacterial
phylum identified in the bottom sediment in the current study was Bacteroidetes, with
predominance in winter (c.a. 2.50–17.50%). The Bacteroidetes phylum appears to be dom-
inant in the soil environment, where it indicates soil “fatigue” [29], and in human and
animal intestines [30]. These bacteria thrive on their ability to secrete a variety of carbo-
hydrate-active enzymes (CAZymes) that act on the highly diverse glycans in the soil [29].
Importantly, soil Bacteroidetes are less well studied than the human gut symbionts; how-
ever, increasing numbers of studies are exploring the important biochemical and physio-
logical phenomena associated with these bacteria [30].
Betaproteobacteria was the most abundant class of bacteria identified in the current
study (Figure 4). Indeed, its representatives occurred at similar abundance levels in all sea-
sons and in all bottom sediment locations, indicating that they are ubiquitous bacteria [31]
and prefer bottom sediments of water bodies as niches for colonization. In addition, they
are abundantly found in drinking water, including mineral water [32]. They are potential
but sometimes overlooked opportunistic pathogens that can be transmitted through water
and aqueous solutions [33]. In addition, some Betaproteobacteria present in drinking water
with inherent and sometimes acquired antibiotic resistance, carrying virulence factors, and
present in biofilm structures can persist even after water disinfection and reach the con-
sumer [32]. The class Gammaproteobacteria was also recorded in all seasons and all bottom
sediment sampling points (Figure 4). Their presence is ecologically important, as they play
an important role in nutrient cycling in coastal marine ecosystems [33], in addition to being
responsible for carbon fixation in coastal sediments [34] and having metal-reducing abilities
Biology 2022, 11, 913 17 of 27
[35]. The class Deltaproteobacteria was most abundantly recorded in the bottom sediments
from Cardinal Pond during summer (Figure 4); nonetheless, their presence was confirmed
in all seasons. Bacteria of this class are important members of the marine microbiota with
diverse capabilities of reductive dehalogenation, respiration of organohalogens (halogen-
ated compounds that contain at least one halogen (fluorine (F), chlorine (Cl), bromine (Br),
or iodine (I)) bound to carbon), thus playing an important role in the natural cycling of or-
ganohalogens in the environment [36].
The highest bacterial diversity in the bottom sediments from Cardinal Pond was ob-
served at the taxonomic level of genera (Table 1), with the indication that the presence of
individual bacterial genera varied with the seasons (Figure 5). Considering the presence
of all genera identified in the current study, the highest biodiversity of bacteria in the
bottom sediments was shown in autumn and winter (Table A1). Some genera noted in
autumn were not present in other seasons of the year, i.e., Aeromonas, Arthrobacter, Aci-
netobacter, Polaromonas, Methylobacter, Flavobacterium, etc., whereas the presence of, e.g.,
Shewanella and Iodobacter was detected only in winter (Table A1). In terms of the sediment
collection points, most genera in all seasons were identified in sediments Sed_1, Sed_5,
Sed_7, Sed_9, and Sed_10.
Table 1. Seasonal changes in the number of dominant bacteria in the bottom sediments based on
taxonomic affiliation: phylum-class-genus.
Seasonal Variation in Abundance
Main Dominants
Phyla 13 14 12 15
Classes 28 28 22 29
Table A1 (Appendix A)
In general, Thiobacillus was the undisputed generic dominant in the bottom sedi-
ments, regardless of the season. Its contribution to the bacterial structure was significant
at most of the analyzed measurement points in Cardinal Pond. Gp3, Gp13, Gp18 domi-
nated in summer, likewise in autumn, together with representatives of Polaromonas, Fla-
vobacterium, and Thermoanaerobaculum. In turn, Flavobacterium, Gp3, and Gp13 occurred
most frequently in winter and in the greatest number of locations (Table A1). All these
bacteria are ecologically important genera, i.e., Thiobacillus, which is widespread in marine
and terrestrial habitats, oxidizes sulfur, producing sulfates and generates sulfuric acid in
deep ground deposits that dissolve metals in mines [37]. Polaromonas are among the dom-
inant bacteria of glacial ice and sediments worldwide [38] with the ability to oxidize a
wide array of unusual energy sources, including H2 [39], arsenite [40], and a broad range
of recalcitrant organic compounds [38]. With these characteristics, Polaromonas are re-
ferred to as metabolically diverse “opportunitrophs” [41] that take advantage of transient
periods of higher temperatures and substrate availability occurring in extreme environ-
ments. Flavobacterium and Thermoanaerobaculum are able to hydrolyze a wide variety of
organic compounds, e.g., several carbohydrates and biomacromolecules [42], thus their
presence in bottom sediments is desirable. Acidobacteria subgroups Gp3, Gp13, and Gp18
are usually the best known for the most positive correlations with gene families associated
with carbon degradation, especially those involved in hemicellulose degradation [43].
The analysis carried out with the use of the Biolog EcoPlate system evidenced similar
trends as NGS (with respect to biodiversity) in the variability of metabolic activities under
Biology 2022, 11, 913 18 of 27
the influence of the season, which indicates different dynamics of microbiological activity
in the sediments during the year. By combining both analytical techniques, it was possible
to identify the highest and lowest catabolic activity and changes in the structure of the
bacterial microbiome in the bottom sediments from the culture pond. AWCD and H
reached the highest level in summer, followed by winter, autumn, and spring, which
clearly indicates the highest peak of metabolic activity and biodiversity in summer and
winter, while the beginning of the growing season (spring) is the time of the lowest mi-
crobial activity in the bottom sediments. The analysis of the preferences of the microor-
ganisms present in the bottom sediments for utilization of carbon sources revealed that
carboxylic and acetic acids and carbohydrates were metabolized most readily (in about
29–30%), followed by amino acids (about 20%), while amines and amides were metabo-
lized the least readily (in about 7%).
Only a few studies on the metabolic activity of bottom sediments performed using the
Biolog EcoPlate system are currently available, hence the results presented in this paper ex-
pand the knowledge in this area, as they were obtained through year-long monitoring of
the bacterial activity. Zhao et al. [44] studied the functional diversity of the bacterial micro-
biome in water and sediment from shrimp ponds and found that the average value of ab-
sorption of the carbon sources utilized by the microorganisms in the sediment was signifi-
cantly higher than that found in the water samples. The researchers [44] reported an H value
in the sediment samples in the range of 3.015–3.28, which is in agreement with our observa-
tions and the H value of 3.16–3.34. However, preferences for carbon utilization by microor-
ganisms reported by Zhao et al. were different from our findings, as they noted that amino
acids and polymers were the most preferable carbon sources [44]. In turn, similar patterns
in carbon utilization to those noted in the current study were evidenced in a horizontal sub-
surface-flow constructed wetland, where higher utilization of carbohydrates, carboxylic ac-
ids, and amino acids was noted in upper front substrate microorganisms than in lower back
substrate microorganisms [45]. Additionally, Wu et al. [46] observed that microorganisms
originating from bottom sediments of the freshwater lake Taihu in China metabolized car-
boxylic and acetic acids and carbohydrates most frequently after polymers. It was also noted
that the sediment microbial communities of lakes in summer and autumn showed more
versatile substrate utilization patterns than in spring [47], which was revealed in the current
study. In general, the lake and river sediment microbiome in summer and autumn preferred
to use carbohydrates [46], likewise in our study.
The bacterial functional analysis performed with the use of the KEGG tool confirmed that
aerobic respiration I with the use of cytochrome C was the main pathway used by the micro-
biome of the studied bottom sediments. In this context, Hamada et al. [48] revealed that cbb3
oxidases (cytochrome C), which are commonly known as aerobic respiratory enzymes, were
involved in denitrification and influenced the lifestyle of Psedomonas aeruginosa PAO1 in an-
oxic conditions. We evidenced that the investigated bottom sediments from Cardinal Pond
were characterized by the highest relative abundance of bacteria capable of using CoQ10 path-
ways. George et al. [49] indicated that most Gram-negative facultative anaerobes use CoQ10
pathways, and these pathways can be identified in some anoxygenic phototrophic bacteria
(isolated from lake sediment). Additionally, the presence of Gram-negative facultative anaer-
obes may correlate with temporary flooding and drying of the pond [49].
5. Conclusions
The combination of two research techniques (NGS and CLPP) resulted in the identi-
fication of changes in the structure of bacteria inhabiting bottom sediments and their func-
tional activity under the influence of seasons. Thus, the study provided new knowledge
of the still rather poorly explored microbiome of bottom sediments. Both the location of
sampling in Cardinal Pond and the season of the year had an impact on its biodiversity
and metabolic activity. Proteobacteria, Acidobacteria, and Bacteroidetes were identified
as dominant phyla, followed by Euryarchaeota, Firmicutes, Ignavibacteriae, Campilobac-
terota, Chloroflexi, and Actinobacteria. Beta- and Gammaproteobacteria were the most
Biology 2022, 11, 913 19 of 27
abundantly represented classes, but Delta- and Alphaproteobacteria, Acidobacteria-Gp3,
Acidobacteria-Gp13, and Acidobacteria-Gp18 were also present with fairly high fre-
quency, depending on the season. At the taxonomic level of genera, the following repre-
sentatives dominated: Thiobacillus and Gp3 in spring, Thiobacillus, Paenibacillus, Gp3, and
Gp13 in summer, Thiobacillus, Gp3, Aeromonas, Arthrobacter, and Pseudomonas in autumn
and Thiobacillus, Gp3, Gp13, Flavobacterium, and Shewanella in winter. Compared to the
other seasons, the greatest modifications of the bacterial microbiome structure were ob-
served in summer. Similarly, the highest metabolic activity of bacteria inhabiting bottom
sediments was found in summer. It was expressed by the AWCD index, which reached
the following gradient: summer > winter > autumn > spring (analogically to the H index).
The microbial preferences for the utilization of carbon sources were as follows: carboxylic
and acetic acids > carbohydrates > amino acids > polymers > amines and amides. Aerobic
respiration I with the use of cytochrome C was the main pathway used by the microbiome
of the studied bottom sediments.
Supplementary Materials: The following supporting information can be downloaded at:, Figure S1: Metastat analysis showing
significant differences in bacteria composition (class level) in smples from summer compared to
other analyzed sea, Figure S2: Metastat analysis showing significant differences in bacteria compo-
sition (genus level) in samples from summer compared to other analyzed sea, Table S1: Sequencing
data quality, Table S2: Diversity indices (NGS), Table S3: Basic characteristic of bottom sediments.
Author Contributions: Conceptualization, A.W.; methodology, A.G., K.S., A.M.-G., and A.K. (Ag-
nieszka Kuźniar); software, J.G., A.M.-G., and A.G.; validation, J.G., A.M.-G., A.K. (Anna
Kruczyńska), A.W., and A.K. (Agnieszka Kuźniar); formal analysis, A.K. (Agnieszka Kuźniar), A.G.,
A.M.-G., and J.G.; investigation, A.G., K.S., and A.K. (Anna Kruczyńska); writingoriginal draft
preparation, A.W.; writingreview and editing, A.G. and A.K. (Agnieszka Kuźniar); visualization,
A.K. (Anna Kruczyńska), A.M.-G., and A.G.; supervision, A.G.; funding acquisition, A.W. All au-
thors have read and agreed to the published version of the manuscript.
Funding: The APC was funded by the John Paul II Catholic University of Lublin.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Acknowledgments: The authors thank Włodzimierz Rybacki, Artur Banach, and Anna
Sochaczewska for their help in the bottom sediment sampling and the management board of CGFP
sp. z o.o. (Jacek Podlewski) for providing the opportunity to conduct the research at Cardinal Pond.
Conflicts of Interest: The authors declare no conflicts of interest.
Biology 2022, 11, 913 20 of 27
Appendix A
Biology 2022, 11, 913 21 of 27
Figure A1. Heat maps illustrating seasonal changes in the unclassified genera of bacteria in the
selected location of the bottom sediments in Cardinal Pond (1–10 Sedsediment sampling points;
Spspring, Susummer, Aautumn, WWinter).
Biology 2022, 11, 913 22 of 27
Biology 2022, 11, 913 23 of 27
Figure A2. Heat maps of carbon utilization patterns of 31 substrates from the Biolog EcoPlates in-
cubated for 120 h with the bottom sediments in Cardinal Pond. The results are visualized as stand-
ardized data of absorbance measurement at a wavelength of 590 nm (higher values mean higher
functional activity).
Biology 2022, 11, 913 24 of 27
Table A1. Genus-classified bacteria present in the bottom sediments and their seasonal variation (+
indicates the presence of a particular genus).
Biology 2022, 11, 913 25 of 27
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