Refine
Year of publication
- 2025 (1)
Document Type
- Doctoral Thesis (1)
Language
- English (1)
Has Fulltext
- yes (1)
Is part of the Bibliography
- no (1)
Keywords
- 16S rRNA amplicon sequencing (1)
- 16S rRNA-Amplikon-Sequenzierung (1)
- Abwasser (1)
- Activated sludge (1)
- Belebtschlamm (1)
- Biotransformation (1)
- Microbial community (1)
- Micropollutants (1)
- Mikrobielle Gemeinschaft (1)
- Process conditions (1)
Water is the basis of all life, a biotope for a variety of organisms and an important component of the
natural balance. For this reason, it is essential to protect water from contamination by anthropogenic
organic micropollutants (MPs) and to develop various innovative strategies for the treatment and reuse
of wastewater. Conventionally biologically treated municipal wastewater contains many organic MPs
that pose a potential threat to aquatic ecosystems and drinking water resources. To date, the focus has
been on physicochemical processes such as activated carbon treatment or ozonation to improve the
removal of MPs in wastewater treatment plants (WWTPs), while knowledge of biological removal
processes and ways to optimize the biological removal of organic MPs is comparatively limited.
Especially in arid and semi-arid regions, treated wastewater is also an important resource for wastewater
reuse for irrigation or drinking water treatment. In consequence of increasing droughts due to climate
change, the reuse of treated wastewater is also increasingly being discussed in Germany. Often the water
is reused after targeted groundwater recharge, while concepts for optimizing the removal of MPs during
soil passages are still scare. In order to assess the potential and limitations of biological wastewater
treatment as well as targeted groundwater recharge for the removal of organic MPs, a better knowledge
of the biological biotransformation processes and the environmental conditions influencing them is
required. Microbial communities and their enzyme pools play a key role in these processes. However,
the influence of environmental conditions on the composition and functional characteristics of microbial
communities, and how this in turn affects the biotransformation potential of MPs with different
structural characteristics, has been little studied. In addition, knowledge about MP-biotransforming
bacteria is often derived from enrichment or even pure culture studies, which are not directly transferable
to the environment.
Against this background, this dissertation focuses on the systematic investigation of the
biotransformation of MPs in contact with activated sludge (simulation of biotransformation processes
in biological treatment stages of WWTPs) and filter materials (simulation of processes in groundwater
recharge systems) under defined laboratory conditions. The aim is to elucidate relationships between
the composition and functional characteristics of microbial communities, microbiologically determined
biotransformation processes and their optimal process conditions, as well as the chemical structure and
primary enzymatically catalyzed biotransformation reactions of MPs.
The first part of the work focuses on the composition of microbial communities and the
biotransformation of MPs depending on defined process parameters in five differently operated
wastewater reactors, divided into two reactor cascades at pilot scale and one full-scale WWTP. The first
cascade consisted of three reactors, the first of which served as a reference reactor with conventional
activated sludge (CAS) treatment. The following reactors operate under anoxic to anaerobic and strictly
anaerobic conditions, respectively. The other cascade consists of two reactors, the first anaerobic and the second aerobic, followed by a simplified vessel to prevent nitrate output, as half of the effluent was
recirculated to the first anaerobic reactor. All five reactors and the WWTP were analyzed for the
biotransformation of 33 MPs and the composition of the microbial community by LC-MS/MS and 16S
rRNA gene sequencing, respectively. The results showed a slight but significant improvement in the
overall biotransformation of MPs in the reactor cascades (about 20%) compared to the WWTP. In
particular, the biotransformation of compounds that are not or only slightly degradable (< 30%) in
conventional wastewater treatment, such as diatrizoate, venlafaxine and diclofenac, was significantly
improved (about 70%). Twelve of the 33 MPs showed an increase in biotransformation of at least 30%
compared to the reference reactor and the WWTP. In detail, the reactor cascade consisting of the
anaerobic pre-treatment showed three times more MPs than the reactor cascade with the anaerobic post-
treatment. Although the environmental conditions (nutrient availability and redox conditions) differed
considerably between the reactors, molecular analysis of the microbial community revealed a core
community of 143 genera, with 54% of the taxa belonging to the phylum Proteobacteria, with the beta
subdivision as the most dominant class. On the other hand, a specialized community consisting of 90
genera was identified that contributed most to the differences between the reactor communities. These
genera were shown to reflect the prevailing nutrient, redox and operating conditions of each treatment.
It was also found that the relative abundances of several genera of the specialized community correlated
with the biotransformation of certain MPs as well as with process parameters (especially redox
conditions). These genera may not necessarily be directly involved in the biotransformation of MPs, but
could be promising biological indicators for the establishment and control of operating conditions
favorable to the biotransformation of certain MPs. For the two reactor cascades analyzed, it was
concluded that the redox conditions as well as the carbon supply were essential factors for the
composition of the specialized community and the biotransformation of the MPs. In addition, the 16S
rRNA gene amplicon sequencing proved to be a complementary tool to confirm process conditions by
correlation. In conclusion, the efficiency of MP biotransformation in conventional WWTPs depends on
key parameters such as redox conditions, biodiversity or the presence of several genera of specialized
microbial communities identified as indicator organisms.
Against the background of previous knowledge that biofilms from moving bed biofilm reactors
(MBBRs), which have been used so far in a few WWTPs, are more efficient in biotransforming certain
MPs, the second study used laboratory batch experiments to investigate the extent to which the
biotransformation potential of carrier-attached biofilms and suspended sludge from hybrid MBBRs
differ between three WWTPs, and whether these differences were also reflected in the composition of
the bacterial communities. The 31 MPs investigated were grouped according to their biotransformation
rates and examined for relationships between the biotransformation behavior and the known primary
biotransformation reactions are discernible. In general, the study confirmed the potential of hybrid
MBBRs for improved biotransformation of a variety of MPs. This could be attributed to an increased
biotransformation potential of the carrier-attached biofilms, especially for oxidatively degradable substances such as trimethoprim, diclofenac and mecoprop. Correlation analyses showed statistically
significant relationships between the occurrence of certain bacterial genera (e.g. Acidibacter, Nitrospira
or Rhizomicrobium) and the biotransformation rates of certain MPs. Thus, some of the identified genera
were also discussed as suitable indicators for the biotransformation potential of suspended sludge or
carrier-attached biofilm.
In the third part of the work, the biotransformation of up to 78 MPs was investigated under different
redox and substrate conditions in a defined column system at laboratory scale as well as ex situ under
uniform incubation conditions by incubating the column filter material in batch experiments. MPs were
categorized based on their biotransformation behavior and how well these categories matched primary
transformation reactions known from the literature or predicted using the Eawag pathway prediction
system for modelling microbial biotransformation pathways. The column system investigated consisted
of two large columns filled with technical sand, an intermediate aeration and four smaller columns, all
connected in series. In the first column, characterized by a carbon-rich environment with high biomass
and strong oxygen depletion, 23 MPs were efficiently removed (>80% removal), while 19 substances
were better or exclusively removed under carbon-limited oligotrophic conditions in the columns after
re-aeration. While the MPs removed predominantly in the first column were mostly attributed to
biotransformation by comparatively ubiquitous and fast transformation reactions such as the oxidation
of alcohols or amide hydrolysis, the biotransformation of MPs removed more efficiently under the oxic
and oligotrophic conditions in the rear columns was characterized by slower transformation reactions
such as N-dealkylation of primary and secondary amines or the hydroxylation of aromatic rings. In
addition, several specific reactions described in the literature, such as the cleavage of C-Cl and C-O
bonds, could only be identified under oligotrophic conditions. The results suggest that conditions of
limited carbon availability may favor the development of alternative metabolic biotransformation
pathways. Furthermore, the observed association between system-specific MP removal and the expected
primary biotransformation reactions from the literature and prediction systems could serve as a basis for
accurately identifying the relevant enzymes in future studies, using metagenomic or metatranscriptomic
data.
Based on these considerations, molecular biological investigations were carried out in the fourth part of
this work by generating sequence-based data complementary to the biotransformation rate constants
during the batch experiments carried out in the third study. A first aim was to identify a statistical relation
between the microbial composition, the process conditions and the nutrient availability as well as the
biotransformation potential of 42 MPs depending on the biomass in the columns of the system.
Furthermore, the metabolic activities of the microbial communities of the first two columns of the
system were analyzed and the identified active enzymes were assigned to known metabolic pathways
and the activities were compared. In addition, the extent to which the biotransformation potential of the
columns was also reflected in the activity of enzymes that have been proven to belong to biotransformation pathways of MPs was investigated. To this end, modern sequencing methods were
used to generate data on the composition of the microbial community (16S rRNA gene sequencing) and
its actual activity in the form of expressed genes (metatranscriptome sequencing). The biotransformation
rates from the batch experiments of the third study were used by normalizing them with the gene copy
numbers (kbio), which were representative of the biomass. For half of the MPs, the highest normalized
kbio values were identified in the rear columns with increased carbon-limiting conditions. Furthermore,
most of the MPs investigated, such as sulfathiazole, sulfamethoxazole or rufinamide, showed an
improved biotransformation potential in these columns. The higher biotransformation potential under
carbon-limiting conditions was also confirmed by metatranscriptomic analysis, where an increased
metabolic capability and a higher functional activity to degrade MPs were observed in the carbon-
limiting column compared to the first nutrient-rich column. Taxonomic analysis revealed a decreasing
trend in biodiversity with a simultaneous increase in carbon-limiting conditions. Furthermore, the
microbial community differed depending on the nutrient and process conditions of the column materials
analyzed. However, within the carbon-limiting columns, the microbial community was similar and were
dominated by the genus Pseudomonas. By relating the biotransformation of MPs to the relative
abundance of taxa in the carbon-limiting columns, the genera of the phylum Acidobacteria and the
classes Alpha- and Gammaproteobacteria showed particularly high associations. In addition, four genera
showed a statistically positive correlation with the two structurally similar MPs sulfamethoxazole and
sulfathiazole.
Overall, no general improvement in biotransformation could be found for all MPs under the conditions
considered, with system- and condition-specific changes observed for individual compounds. However,
the studies indicated favorable environmental conditions for groups of MPs that showed increased
biotransformation linked with high abundant taxa, especially under carbon-limiting conditions and in
carrier-attached biofilms.