Wastewater Bacteria (Wastewater Microbiology)
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When the freshwater microalga Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense were deployed as free suspensions in unsterile, municipal wastewater for tertiary wastewater treatment, their population was significantly lower compared with their populations in sterile wastewater. At the same time, the numbers of natural microfauna and wastewater bacteria increased. Immobilization of C. sorokiniana and A. brasilense in small (2-4 mm in diameter), polymer Ca-alginate beads significantly enhanced their populations when these beads were suspended in normal wastewater. All microbial populations within and on the surface of the beads were evaluated by quantitative fluorescence in situ hybridization combined with scanning electron microscopy and direct measurements. Submerging immobilizing beads in wastewater created the following sequence of events: (a) a biofilm composed of wastewater bacteria and A. brasilense was created on the surface of the beads, (b) the bead inhibited penetration of outside organisms into the beads, (c) the bead inhibited liberation of the immobilized microorganisms into the wastewater, and (d) permitted an uninterrupted reduction of ammonium and phosphorus from the wastewater. This study demonstrated that wastewater microbial populations are responsible for decreasing populations of biological agents used for wastewater treatment and immobilization in alginate beads provided a protective environment for these agents to carry out uninterrupted tertiary wastewater treatment.
Conventional detection of microorganisms is still based on their cultivation and is not well suited for the analysis of complex samples such as wastewater. Studies demonstrate that up to 99.9% of all bacteria in wastewater cannot be cultivated. But even simple staining methods are not sufficient due to their lack of specificity and the morphovariability or gram variability of the bacteria. In order to obtain a realistic picture of the microbiological conditions in a wastewater treatment plant, it is necessary to analyze the bacteria directly in the sample and without any detours.
Microorganisms in urban sanitary sewers exhibit community properties that suggest sewers are a novel ecosystem. Sewer microorganisms present both an opportunity as a control point for wastewater treatment and a risk to human health. If treatment processes are to be improved and health risks quantified, then it is necessary to understand microbial distributions and dynamics within this community. Here, we use 16S rRNA gene sequencing to characterize raw influent wastewater bacterial communities in a 5-year time series from two wastewater treatment plants in Milwaukee, WI; influent wastewater from 77 treatment plants across the USA; and wastewater in 12 Milwaukee residential sewers.
In Milwaukee, we find that in transit from residences to treatment plants, the human bacterial component of wastewater decreases in proportion and exhibits stochastic temporal variation. In contrast, the resident sewer community increases in abundance during transit and cycles seasonally according to changes in wastewater temperature. The result is a bacterial community that assembles into two distinct community states each year according to the extremes in wastewater temperature. Wastewater bacterial communities from other northern US cities follow temporal trends that mirror those in Milwaukee, but southern US cities have distinct community compositions and differ in their seasonal patterns.
Urban sewers collect wastewater from a variety of sources, including stormwater, industrial waste, and residential sewage. Sewer pipes transport wastewater to wastewater treatment plants (WWTPs), where nutrients and microorganisms are removed and select microorganisms are cultivated to aid treatment processes [1]. Imbalanced WWTP microbial communities can disrupt treatment and create challenging and costly problems. For instance, WWTPs typically settle activated sludge to separate it from treated wastewater, but overgrowth of filamentous bacteria causes poor settling, which deteriorates effluent quality and may require significant process alterations to remedy [2]. The goal of wastewater treatment is to foster beneficial microbial communities and remove problematic ones, and WWTP influent can be a source of each [3,4,5].
Sewers serve as more than conveyance for wastewater. The consistency in sanitary sewer microbial community composition suggests that sewers represent a recently formed ecosystem [6]. Some resident sewer microbes induce pipe corrosion [7, 8], display pathogenic lifestyles [9], or propagate antibiotic resistance genes [10, 11], including those that survive treatment and persist in receiving waters [12,13,14,15]. Aging and inadequate infrastructure also introduces sewer bacteria to the environment by leaching wastewater through corroded pipes [16,17,18] or through deliberate release during sewer overflows [19, 20]. Sewage discharge regularly impairs recreational waters, causes coastal beach closures, and poses a significant risk to human health [21]. Despite the potential importance of resident sewer bacteria, there is not a thorough understanding of whether the majority of these microorganisms exhibit predictable abundance patterns through time or among sewer systems, partition to various substrates in wastewater, or survive for prolonged periods in natural aquatic systems after discharge.
Many aquatic ecosystems undergo seasonal changes that drive biological change, which in turn creates repeating and predictable microbial community structures and ecosystem services [22,23,24,25]. As sewers are a primarily aquatic environment, it is possible the resident microbial communities also exhibit temporal community assembly patterns. Initial studies suggest this may be the case. Guo et al. [26] revealed diurnal trends in WWTP influent microbial communities that were driven by change in flow rate between day and night, where low flow resulted in less sloughing of pipe bacteria and thus a change in composition. Although this study provided evidence of repeatable microbial dynamics, these dynamics were driven by short-term physical factors. To the best of our knowledge, no study has analyzed whether pre-treatment wastewater microbial communities are also impacted by longer-term changes (months or years) to their environment. Uncovering patterns of assembly by sewer microbial communities will aid in designing models to predict wastewater composition, enable targeted treatments for microorganisms of interest, and identify whether temporal community variation relates to altered human and/or environmental health risks from untreated discharge.
Forward and reverse reads were quality-filtered using FastQC [29] and primers were trimmed with Cutadapt [30]. We processed the three wastewater datasets simultaneously with the R package DADA2 [31], following the protocol at , with the following exceptions: during filtering, reads were truncated at 230 bp, and reads with quality scores lower than 10 were removed; after merging, sequences were removed that did not have lengths within 5% (355 to 393) of the median sequence length (374 bp). Taxonomy was assigned to resulting amplicon sequence variations (ASVs) using Silva v. 132 [32]. ASVs that were not classified as bacteria or were classified as mitochondria or chloroplasts were removed. Contaminant ASVs from the mock community and negative control were identified with the R package Decontam [33] and subsequently removed.
a Alpha diversity (measured with Shannon diversity), and b beta diversity (measured with Bray-Curtis dissimilarity) in raw wastewater bacterial communities. Diversity was measured in wastewater treatment plant influent in the 5-year time series of JI, in Milwaukee, WI; in JI and SS in Milwaukee, WI; from 77 WWTPs across the USA; and in wastewater collected from residential Milwaukee neighborhood sewers. Boxes depict the median and first and third quartiles. Whisker lines extend to interquartile ranges × 1.5 and points are outlier values
The majority of wastewater bacteria were not associated with the human microbiome (Fig. 2c). In residential sewer communities, 35.9 ± 7.5% of reads belonged to ASVs attributed to the human microbiome, but in the 5-year time series of two Milwaukee WWTPs, the proportion dropped to 11.0 ± 2.8%. Similarly, across the USA, only 12.4 ± 5.7% of reads were human-associated. Of the human microbiome sources, stool was the greatest contributor of ASVs to WWTP influent (9.0 ± 4.7%; Fig. 2d). Overall, we find that the majority of reads in wastewater were assigned to ASVs that were not associated with the human microbiome (88.0 ± 5.0%), and we considered them to be sewer-associated for subsequent analyses.
a Principal coordinate analysis (PCoA) of influent bacterial communities from two Milwaukee WWTPs sampled once a month for 5 years. Points indicate influent bacterial community samples, color denotes the month sampled, and shape indicates the source WWTP. Axis 1 is set as the y-axis for visualization purposes. b PCoA Axis 1 scores from both WWTPs over time (solid grey lines) plotted with wastewater temperatures (blue dashed lines)
Seasonal bacterial community variation was driven more by abundance changes of common sewer-associated ASVs than by human-associated ASVs. Dendrogram clustering of normalized sewer-associated ASV abundances (Milwaukee time-series) illustrated that many common wastewater bacteria (e.g., Acinetobacter, Arcobacter, Cloacibacterium, Flavobacterium, Lactococcus) exhibited repeating temporal patterns of high/low or low/high abundance in the spring and fall states (Fig. 4). In contrast to the seasonal abundance pattern clustering of sewer-associated ASVs in the influent samples, common human ASVs exhibited less dramatic temporal fluctuations, and these changes were not predictable temporally or with the wastewater environmental data. Instead, human ASV relative abundance patterns often clustered by taxonomic affiliation (Fig. 4). 781b155fdc