HIGH-THROUGHPUT 16S rRNA GENE SEQUENCING REVEALS INTERACTIONS BETWEEN MULTIPLE STRESSORS ON RIVER BIOFILMS UNDER CONTROLLED CONDITIONS Ferran ROMERO*†1, Vicenç ACUÑA1, José Luís BALCÁZAR1, Carme FONT1, Sergi SABATER1,2 † Corresponding
author
* Presenting author.
1Catalan
Institute for Water Research (ICRA), Carrer Emili Grahit 101, 17003 Girona, Spain. 2Institute of Aquatic Ecology, University of Girona, Campus Montilivi, 17071 Girona, Spain.
BACKGROUND Disentangling the individual and interactive effects of multiple stressors on microbial communities is a key challenge to our understanding and management of ecosystems. Next-generation sequencing (NGS) methods based on sequencing of bacterial 16S rRNA gene represent a robust, relatively cheap, fast and almost universal tool for in-depth analysis of changes in community composition after exposure to stress. NGS enables identification of present microbial taxa with high resolution, allowing more complex taxonomic analysis.
Agriculture
Here we summarize two manipulative studies aimed at developing mechanistic understanding on the effects of multiple stressors on river biofilms. Artificial channels (n = 24) were used in replicated designs and realistic stress levels were applied during 30 days to biofilms growing on coarse and fine sediment samples. After that, biofilm was removed from sediment, DNA was extracted and 16S rRNA was sequenced using Illumina sequencing technology.
Climate Change Urban runoff
Multi-stress context
Results indicate strong effect of desiccation on bacterial community composition, particularly on coarse sediment, whereas fine sediment was more affected by temperature. Pesticides had little effect, but WWTP effluent (particularly at > 50 % of concentration) had strong effects. The strongest interaction took place between WWTP effluent and sediment type, although temperature and pesticides/desiccation also resulted in significant interactions.
Bacterial community response River biofilm
EXPERIMENTAL DESIGN EXPERIMENTAL CHANNELS
Experiment 1
Stressor
Levels
Desiccation (D)
7-day complete desiccation
Urban WWTP effluent (We)
Gradient (from 0 % to 100 % of the total flow)
RESPONSE OF BACTERIAL COMMUNITY
Effects of single and combined stressors
Threshold Indicator Taxa Analysis (M)ANOVAs
Experiment 2
Stressor
Levels
Temperature (T)
18.5°C vs. 20°C
Pesticides (P)
EXPERIMENT 1. THE EFFECTS OF URBAN POLLUTION ARE MODULATED BY DESICCATION.
COARSE SEDIMENT (% of total sequences, most abundant classes)
FINE SEDIMENT (% of total sequences, most abundant classes)
40 20
0%
14%
29% 43%
58% 72% 86% 100%
80 60 40 20
0%
14%
29% 43%
58% 72% 86% 100%
Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Deltaproteobacteria Proteobacteria UAC Bacilli Planctomycetia Cyanobacteria UAC Saprospirae Cytophagia Verrucomicrobiae Synechococcophycideae SJA-4 (phylum TM6) Bacteria UAC Bacteroidetes Flavobacteriia Clostridia Other
80 60 40 20
CT
D
T
P
DP
TD
TP
TDP
80 60 40 20
CT
D
T
P
DP
TD
TP
TDP
% of Wastewater in the treatment
% of Wastewater in the treatment
Single stressor, effect size PERMANOVA F-value WWTP effluent (We) 5.19 - 8.11 Desiccation (D) 1.42 - 5.44 Sediment type (S) 2.96 - 4.36 Multiple stressor, effect size PERMANOVA F-value We * D We * S D*S We * D * S
% of total sequences
60
FINE SEDIMENT (% of total sequences, main classes)
COARSE SEDIMENT (% of total sequences, main classes)
W D W D W D W D W D W D W D W D c__Alphaproteobacteria c__Gammaproteobacteria c__Betaproteobacteria p__Proteobacteria_unclassified c__Deltaproteobacteria c__Planctomycetia c__Saprospirae c__Cytophagia c__Flavobacteriia c__Verrucomicrobiae p__Verrucomicrobia_unclassified c__Spartobacteria c__Acidobacteria-6 c__Chloracidobacteria c__Actinobacteria c__Clostridia c__Oscillatoriophycideae c__Anaerolineae c__Ellin6529 k__Bacteria_unclassified
% of total sequences
% of total sequences
W D W D W D W D W D W D W D W D 80
EXPERIMENT 2. AGRICULTURAL CHEMICALS INTERACT WITH CLIMATE CHANGE-RELATED STRESSORS.
% of total sequences
Desiccation (D)
90 mL s-1 vs. 10mL s-1 (no water column) Absence vs. Presence
not significant
9.62 - 15.88 not significant
1.74 - 2.25
The effect of WWTP effluent was similar across taxonomic levels, whereas lower taxonomic levels showed stronger sensitivity to desiccation. As retrieved from Threshold Indicator Taxa Analysis (TITAN), the shift in bacterial community composition was particularly important at > 50 % of WWTP effluent. Desiccation involved changes in bacterial community composition, favoring soil-associated members of Alphaproteobacteria (e.g. Aminobacter, Kaistia, Sphingobium).
Single stressor, effect size PERMANOVA F-value Temperature (T) 1.05 - 3.44 Desiccation (D) 1.43 - 18.18 Pesticides (P) 0.13 - 3.67
T*D T*P D*P T*P*D
Multiple stressor, effect size PERMANOVA F-value 0.84 - 3.74 0.24 - 2.64 not significant not significant
The effect of desiccation was particularly strong for coarse sediment communities, while fine sediment was more affected by increased temperature. Pesticides had little to no effect. Overall, stress conditions seemed to increase the abundance of Proteobacteria and Firmicutes, while decreasing other phyla (i.e. Bacteroidetes and Planctomycetes). Many bacterial taxa were affected by interactive effects, particularly T * P and T * D. Lower taxonomic levels (i.e. genus) were more sensitive to interactions between stressors.
CONTACT
ACKNOWLEDGEMENT
Name: Ferran Romero Blanch Email:
[email protected] Web: www.globaqua-project.eu
The research leading to GLOBAQUA has received funding from the European Union’s Seventh Programme under grant agreement No 603629. Authors also thank the organizers of the 5th Biofilm Workshop for the grant to Ferran Romero.
