Bacterial Structure and Community-Level Physiological Profiles in Water From Vistula River, Lubelskie, Poland

: The Vistula River is Poland's longest river, and its section called the Lesser Poland Gorge of the Vistula (Polish: Małopolski Przełom Wisły) belongs to two Natura 2000 Areas. The waters of the Vistula River periodically flood neighbouring areas during winter or summer floods. In this study we decided to determine the composition of the bacterial community and its metabolic potential in Vistula River water. In order to analyse the structure of bacterial communities, the next generation V3/V4 16S rDNA region sequencing (NGS) using Miseq (Ilumina) and the community-level physiolo gical profiles (CLPP) method using the Biolog® EcoPlate™ system were applied. A total of 413 operational taxonomic units (OTU) were obtained, of which 377 could not be classified at genera level. Among those identified, Arenimonas (2.13%), Brevundimonas (1.44%) and Flavobacterium (1.05%) predominated. The most intensively degraded group of substrates were carbohydrates (30.93%) and the least amines and amides (6.44%). The NGS analysis did not indicate a sanitary risk to humans. A large number of unclassified bacteria indicate a still low level of knowledge and limitations concerning the world of bacteria.


Introduction
The Vistula River is Poland's longest river, and its section called the Lesser Poland Gorge of the Vistula (Polish: Małopolski Przełom Wisły) belongs to two Natura 2000 Areas [1].The waters of the Vistula River periodically flood neighbouring areas during winter or summer floods [2].Among studies on the microbiome of river waters, sanitary analyses to control drinking water dominate [3].The most frequently determined bacteria are Escherichia coli and Enterobacter, Citrobacter and Klebsiella.
The aim of this study was to determine the composition of the bacterial community and its metabolic potential in Vistula River water.

Water from the Vistula River
Water was taken from the Vistula River from Janowiec, Lubelskie Voivodeship (51°19′06.8″N21° and 54′53.5″E) in August 2018.The water was taken from the river at a distance of about 27 m from the riverbank into three sterile 50-litre containers (total ∼ 150 L).Selected water parameters are presented in Table 1. 1 Values are the average of three replicates (n = 3); means ± standard deviation (SD).
The water pH was slightly alkaline and was 8.54 and the electrical conductivity (EC) was 370 µ S cm -1 (Table 1).

The colony PCR and next generation sequencing (NGS)
The direct PCR, also known as colony PCR [4] was performed on water from Vistula.All details of the procedure are described in Furtak et al. ( 2020) [5].The resulting product (about 1500 nt) was then used as a template for V3-V4 region sequencing.

The community-level physiological profiles (CLPP)
The diversity of the metabolic potential of the bacterial community from water was determined using the Biolog EcoPlate™ (Biolog Inc., Hayward, CA, USA) system with 31 different carbon sources in three replicates.
The water was applied (without dilution) to the EcoPlate™ plate in a volume of 120 µ l to each well.EcoPlates™ were incubated at 25 °C for 120 h in the OmniLog® ID System multiplate reader (Biolog Inc., Hayward, CA, USA).The intensity of the wells' colour development was determined spectrophotometrically at λ = 590 nm [7] at 24-intervals using a MicroStation ID (Biolog Inc., Hayward, CA, USA) plate reader.
On the basis of the data from Biolog EcoPlate™ (after 120 h of incubation), the average well colour development (AWCD), richness (R), Shannon diversity (H') and evenness (E) indices were calculated following Garland and Mills (1991) [11].Heatmaps were generated on OmniLog units in R software (3.6.0) using the phyloseq package [12].

Structural diversity of the bacterial community
A total of 413 operational taxonomic units (OTU) were obtained, of which 377 could not be classified at genera level (Table 2).Based on this data the calculated Shannon's biodiversity index was 4.94.In the water taken from the Vistula River 53 different bacterial families were identified, the most numerous of which were Planctomycetaceae (18.5%) and Xanthomonadaceae (13.8%).Among the identified classes of bacteria the most abundant were Betaproteobacteria (21.5%).

Metabolic potential of the bacterial community
The highest AWCD and Shannon's diversity index (H') was recorded after 120 hours of incubation of EcoPlate™ (Figure 3).The microorganisms present in the water most intensively decomposed carbohydrate group substrates (30.93%) and the least intensively compounds classified as amines and amides (6.44%) (Table 2).
The NGS analysis did not indicate a sanitary risk to humans.A large number of unclassified bacteria indicate a still low level of knowledge and limitations concerning the world of bacteria.

Figure 1 .
Figure 1.Sequences that occurred in more than 1% in water sample from Vistula River.

Table 1 .
Selected chemical parameters and trace elements contents (mg/L) in water.

Table 2 .
Selected diversity indices obtained for NGS results.

Table 2 .
Indices obtained for EcoPlate™ results and utilization (%) of group of substrates by bacterial community after 120 h incubation.