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Press xenobiotic disturbance favors deterministic assembly with a shift in function and structure of bacterial communities in sludge bioreactors

16 Oct 2020-bioRxiv (Cold Spring Harbor Laboratory)-

TL;DR: Testing the effect of a continuous (press) xenobiotic disturbance on the function, structure, and assembly of bacterial communities within a wastewater treatment system revealed that deterministic assembly prevailed for disturbed bioreactors, while the role of stochastic assembly was stronger for undisturbed reactors.
Abstract: Disturbance is thought to affect community assembly mechanisms, which in turn shape community structure and the overall function of the ecosystem. Here, we tested the effect of a continuous (press) xenobiotic disturbance on the function, structure, and assembly of bacterial communities within a wastewater treatment system. Two sets of four-liter sequencing batch reactors were operated in triplicate with and without the addition of 3-chloroaniline for a period of 132 days, following 58 days of acclimation after inoculation with sludge from a full-scale treatment plant. Temporal dynamics of bacterial community structure were derived from 16S rRNA gene amplicon sequencing. Community function, structure and assembly differed between press disturbed and undisturbed reactors. Temporal partitioning of assembly mechanisms via phylogenetic and non-phylogenetic null modelling analysis revealed that deterministic assembly prevailed for disturbed bioreactors, while the role of stochastic assembly was stronger for undisturbed reactors. Our findings are relevant because research spanning various disturbance types, environments and spatiotemporal scales is needed for a comprehensive understanding of the effects of press disturbances on assembly mechanisms, structure, and function of microbial communities.

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1
Press xenobiotic disturbance favors deterministic assembly with a shift in function and 1
structure of bacterial communities in sludge bioreactors 2
3
Authors: Ezequiel Santillan
1,2§
, Hari Seshan
1,2,†§
, and Stefan Wuertz
1,2,4
* 4
5
Key words: diversity, disturbance, community structure, stochastic assembly, deterministic assembly 6
7
Affiliations: 8
1
Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, 9
637551, Singapore. 10
2
Department of Civil and Environmental Engineering, University of California, Davis, CA 95616, 11
U.S.A. 12
3
School of Civil and Environmental Engineering, Nanyang Technological University, 639798, 13
Singapore. 14
15
16
17
*Correspondence to: Stefan Wuertz, swuertz@ntu.edu.sg 18
§
E.S. and H.S. contributed equally. 19
Current
affiliation: Stantec Australia Pty Ltd, 52 Merivale St, South Brisbane QLD 4101, Australia.
20
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 16, 2020. ; https://doi.org/10.1101/2020.10.15.341966doi: bioRxiv preprint

2
Abstract 21
Disturbance is thought to affect community assembly mechanisms, which in turn shape community 22
structure and the overall function of the ecosystem. Here, we tested the effect of a continuous (press) 23
xenobiotic disturbance on the function, structure, and assembly of bacterial communities within a 24
wastewater treatment system. Two sets of four-liter sequencing batch reactors were operated in 25
triplicate with and without the addition of 3-chloroaniline for a period of 132 days, following 58 days 26
of acclimation after inoculation with sludge from a full-scale treatment plant. Temporal dynamics of 27
bacterial community structure were derived from 16S rRNA gene amplicon sequencing. Community 28
function, structure and assembly differed between press disturbed and undisturbed reactors. Temporal 29
partitioning of assembly mechanisms via phylogenetic and non-phylogenetic null modelling analysis 30
revealed that deterministic assembly prevailed for disturbed bioreactors, while the role of stochastic 31
assembly was stronger for undisturbed reactors. Our findings are relevant because research spanning 32
various disturbance types, environments and spatiotemporal scales is needed for a comprehensive 33
understanding of the effects of press disturbances on assembly mechanisms, structure, and function of 34
microbial communities. 35
Graphical abstract 36
37
38
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 16, 2020. ; https://doi.org/10.1101/2020.10.15.341966doi: bioRxiv preprint

3
Introduction 39
Sludge bioreactors for wastewater treatment are good model systems for microbial ecology 40
studies
1
. These engineered systems involve measurable functions such as the removal of carbon and 41
ammonia that are relevant in practice
2
and entail complex microbial communities in a controlled 42
environment
3
. In nature, microbial communities drive all biogeochemical cycles globally while 43
providing ecosystem functions necessary for all other life forms to exist
4
. The structure of these 44
communities, which can be described in terms of α- and β-diversity metrics, is believed to shape the 45
ecosystem function they provide
5
. Predicting and managing the functions of microbial communities 46
based on a fundamental understanding of their relationship with community structure remains a key 47
challenge for complex microbial systems. 48
Disturbance is thought to have direct impacts on ecosystems by shifting community structure 49
and function
6
. Chloroanilines are intermediate breakdown products from the use of herbicides and 50
pesticides in agriculture
7
, which can be found in rubber, dye, polymer and pharmaceutical industrial 51
wastewater treatment plants
8,9
, but also in soil and natural aqueous environments
10
. These xenobiotic 52
compounds are known to hamper both carbon and nitrogen removal in bioreactors for wastewater 53
treatment
11
, and can represent a disturbance in sludge bioreactor systems. Press disturbances, which 54
inflict a long-term continuous alteration of taxa abundances by changing their environment
12
, are 55
relevant for both microbial ecology and environmental biotechnology as they can lead systems to 56
alternative stable states with different community function and structure
13
. 57
Community assembly processes are fundamentally related to ecosystem function, as these are 58
thought to shape community structure
14
. These processes can be either deterministic or stochastic, 59
often acting in combination to shape patterns of community assembly
15-17
. In ecology, the contribution 60
of assembly mechanisms is usually quantified via null model analyses
18
. Although disturbance is 61
thought to be a main driver of these underlying community assembly mechanisms
19
, a predictive 62
understanding of its effects remains elusive
20
. Disturbance can favour stochastic assembly 63
mechanisms that could lead communities to deviating states of function and structure
21,22
; hence, 64
assessment of its effects demands replicated study designs
23,24
. Further, while several studies have 65
reported patterns of community assembly in engineered microbial systems
25-28
, relatively few have 66
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 16, 2020. ; https://doi.org/10.1101/2020.10.15.341966doi: bioRxiv preprint

4
addressed the effects of disturbance on community assembly, structure and function of such systems, 67
particularly for bioreactors treating wastewater. 68
The objective of this work was to test the effect of a press disturbance on community assembly 69
mechanisms by introducing a xenobiotic compound, 3-chloroaniline (3-CA), in a replicated set of 70
activated sludge bioreactors with a working volume of four liters, representing a mesocosm scale. 71
Based on our findings in a prior reactor study at a microcosm scale
21
, we expected to see a stronger 72
deterministic effect at the disturbed level. Samples were analysed using 16S rRNA gene amplicon 73
sequencing and effluent chemical characterization. Patterns of α- and β-diversity were employed to 74
assess temporal dynamics of community structure. Assembly mechanisms were quantified via two 75
different mathematical null models, one that assessed the phylogenetic turnover for each bioreactor, 76
and another that evaluated the effective bacterial turnover expressed as a proportion of total bacterial 77
diversity
29
across all replicate reactors. 78
Materials and Methods 79
Experimental design 80
Six sequencing batch bioreactors (SBRs) were operated in parallel and fed synthetic wastewater for an 81
acclimation period of 58 d. The inoculum was taken from the aeration tanks of a full-scale wastewater 82
treatment plant (WWTP) in Singapore. On day 0 of acclimation, the freshly sourced inoculum sludge 83
was mixed well in the laboratory and distributed to all six reactors (2 L each). Each was topped up 84
with 2 L of synthetic wastewater, to a working volume of 4 L per reactor. The reactors were run under 85
identical conditions with 12-h cycles as follows: 20 min of feeding, 180 min of anoxic mixing, 440 86
min of aeration and mixing (dissolved oxygen, DO, maintained at 1-2 mg/L using a feedback loop 87
where aeration would commence at 1 L air/min when probes measured DO as below 1 mg/L and 88
aeration would stop when the DO reading was above 2 mg/L), 50 min of settling and 30 min of 89
effluent (supernatant) discharge. Two liters of effluent were discharged at the end of every cycle and 90
replaced with 2 L of synthetic wastewater at the beginning of the next 12-h cycle, resulting in a 91
hydraulic retention time (HRT) of 48 h. The mixed liquor temperature was maintained at 30
C using 92
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 16, 2020. ; https://doi.org/10.1101/2020.10.15.341966doi: bioRxiv preprint

5
water jackets around the reactors and a re-circulating water heater. Solids were removed regularly 93
from the mixed liquor to maintain a solids retention time (SRT) of about 30 d in each reactor. 94
The synthetic wastewater fed to all six reactors during the acclimation period was adapted 95
from Hesselmann et al.
30
and contained the following (mg/L in in each reactor after feeding): sodium 96
acetate (112.5), dextrose (45), yeast extract (67.5), soy peptone (60), meat peptone (60), casein 97
peptone (90), urea (15), ammonium bicarbonate (90), ammonium chloride (169), disodium hydrogen 98
phosphate (720), potassium dihydrogen phosphate (130), calcium chloride dihydrate (10.5) and 99
magnesium sulphate heptahydrate (112.5). The medium also contained 2 mL of the unaltered trace 100
element stock
30
per liter of medium. The first six components contributed to the COD, amounting to 101
about 500 mg/L in the reactor, the next three (ammonium-based) components contributed to the total 102
loading of about 70 mg N/ L, and the phosphates were used to buffer the medium and maintain a pH 103
of around 7.5 to facilitate the nitrification process. 104
After the 58-d acclimation period, the sustained 3-CA input experiment was started and 105
continued for 132 d. At the start of this experiment, three of the acclimated reactors were randomly 106
assigned to the treatment group (press disturbed with 3-CA) and the other three were assigned to the 107
control group (no 3-CA addition, or undisturbed). The cycle conditions and other parameters were 108
kept the same among all six reactors and were identical to the conditions in the acclimation phase. 109
The 3-CA inputs to the treatment reactors started on day 59. The medium used to feed the treatment 110
reactors was slightly altered: the organic constituents (sodium acetate, dextrose, yeast extract, soy 111
peptone, meat peptone and casein peptone) were scaled down by 20%, resulting in a total COD of 400 112
mg/L from these constituents. The remaining 20% of the COD was fed in the form of 3-CA, resulting 113
in a mixed liquor 3-CA concentration of about 70 mg/L, an environmentally relevant level for 114
WWTPs
11
. The three control reactors continued to receive the same 3-CA-free medium that was used 115
during acclimation. 116
To assess the changes in community composition, samples of sludge were taken from all six 117
reactors at five distinct time points, representing five different periods in the progression of the 118
experiment. These five time points were day 0 (the day the reactors were inoculated for acclimation), 119
day 56 (shortly before 3-CA addition to treatment reactors was commenced), day 63 (shortly after 3-120
.CC-BY-NC-ND 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted October 16, 2020. ; https://doi.org/10.1101/2020.10.15.341966doi: bioRxiv preprint

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